Biologically active peptides

ABSTRACT

A peptide or peptide derivative comprising:
     (i) WDLYFEIVW (SEQ ID NO: 1); or   (ii) a variant amino acid sequence comprising one, two, three or four L-amino acid substitutions in WDLYFEIVW (SEQ ID NO: 1); or   (iii) the retro-inverso variant of the peptide or peptide derivative of either one of parts (i) and (ii),
 
wherein said peptide or peptide derivative has procoagulant activity.
   

     A peptide or peptide derivative comprising:
     (i) an amino acid sequence comprising imfwydcye; or   (ii) a variant amino acid sequence comprising one, two, three, four, five or six amino acid substitutions in imfwydcye,
 
wherein said peptide or peptide derivative has procoagulant activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.14/941,163, filed on Nov. 13, 2015 (now issued as U.S. Pat. No.9,598,464), which is a Divisional of U.S. application Ser. No.14/340,231, filed on Jul. 24, 2014 (now issued as U.S. Pat. No.9,206,234), which is a Division of U.S. application Ser. No. 13/238,751,filed on Sep. 21, 2011 (now issued as U.S. Pat. No. 8,822,638), which isa Divisional of U.S. application Ser. No. 12/425,277, filed on Apr. 16,2009 (now issued as U.S. Pat. No. 8,563,688), which claims priority ofU.S. Provisional Application No. 61/009,326, filed on Apr. 17, 2008, andU.S. Provisional Application No. 61/113,055 filed on Nov. 10, 2008, allof which are incorporated by reference herein in their entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM,LISTING APPENDIX SUBMITTED ON A COMPACT DISK

This invention incorporated by reference the Sequence Listing text copysubmitted herewith, which was created on Feb. 25, 2017, entitled008073_5138_US04_Sequence_Listing.txt which is 202 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to low molecular weight peptides withprocoagulant activity for treatment of patients with a deficiency in FV,FVII, FVIII, FX and/or FXI.

BACKGROUND OF THE INVENTION

The blood coagulation cascade involves a series of serine proteaseenzymes (zymogens) and protein cofactors. When required, an inactivezymogen precursor is converted into the active form, which consequentlyconverts the next enzyme in the cascade. It is divided into threedistinct segments: the intrinsic (contact activation), extrinsic (tissuefactor), and common pathways.

In the intrinsic pathway of the cascade, hemophilia is the mostpronounced bleeding disorder, which results in insufficient generationof factor Xa by factor FIX (FIXa)/factor VIIIa (FVIIIa) complex (theintrinsic tenase complex) leading to an insufficient clot formation.Bleeding may then occur spontaneously or following injury.

Hemophilia is an inherited bleeding disorder and two forms ofhemophilia, hemophilia A and B, are known. Hemophilia A is theconsequence of a deficiency of FVIII and is characterized by hemorrhageinto the joints and muscles. FVIII circulates in plasma at a very lowconcentration and is bound non-covalently to von Willebrand factor(vWF). During hemostasis, FVIII is activated by thrombin, separates fromvWF and acts as a cofactor for activated FIXa-mediated FX activation byenhancing the rate of activation.

Patients with less than 1% normal FVIII are considered to have severehemophilia, with 1-5% moderately severe hemophilia, and with more than5% but less than 40% mild hemophilia.

Nowadays the treatment of choice for the management of hemophilia A isreplacement therapy with various plasma derived or recombinant FVIIIconcentrates. Although specific viral-inactivation steps, includingsolvent-detergent treatment or liquid-phase heat treatment, areavailable to inactivate viruses, possible transmission of poorlycharacterized agents (e.g. prions) in plasma derived concentrates isstill an issue discussed in the art.

FVIII is also synthesized as a recombinant protein for therapeutic usein bleeding disorders. Such products have lowered the risk of viralcontamination. There are many recombinant products on the market for thetreatment of hemophilia A. One of these concentrates is the Advate®FVIII composition, which is produced in CHO-cells and manufactured byBaxter Healthcare Corporation. No human or animal plasma proteins areadded in the cell culture process, purification, or final formulation ofthis product.

Although progress in the production of FVIII to ensure purity, efficacyand viral safety has been made over the past decades, some limitationsremain. First of all, severe hemophilia A patients are frequentlyaffected by anti-FVIII inhibitor antibody formation, rendering thetherapy ineffective.

Approximately 30% of patients with severe HA develop alloantibodyinhibitors that can neutralize FVIII (Hay, Haemophilia 2006; 12 Suppl6:23-9; Peerlinck and Hermans, Haemophilia 2006; 12:579-90). Theseinhibitors are typically immunoglobulin G (IgG), predominantly of theIgG4 subclass, that do not fix complement and do not result in theend-organ damage observed with circulating immune complexes. Theinhibitors occur at a young age (about 50% by 10 years), principally inpatients with less than 1% FVIII. Furthermore, acquired hemophilia mayoccur, which is the development of FVIII antibody inhibitors in personswithout a history of FVIII deficiency. This condition can be idiopathic(occurring in people >50 years), it can be associated with collagenvascular disease or the peripartum period, or it may represent a drugreaction (e.g., to penicillin). For clinical purposes, the magnitude ofthe antibody response can be quantified through the performance of afunctional inhibitor assay from which the Bethesda unit (BU) inhibitortiter can be obtained. The International Society of Thrombosis andHaemostasis (ISTH) definition of a high titer response is >5 BUs and itsdefinition of a low titer response is between 0.5 and 5 BUs.

Attempts to overwhelm the inhibitors with large doses of human FVIIIhave been tried. Also porcine FVIII, which has low cross-reactivity withhuman FVIII antibody, has been administered. More frequently,FVIII-bypassing agents, including activated prothrombin complexconcentrates (e.g. FEIBA (Factor Eight Inhibitor Bypassing Agent) andrecombinant activated factor FVII (FVIIa) have also been used.

Because therapeutic polypeptide drugs such as FVIII are also rapidlydegraded by proteolytic enzymes in addition to the drawback of inhibitordevelopment, FVIII needs to be frequently administered intravenously.Taking into account the average half-lives of the various FVIII productsin the circulation, this can usually be achieved by giving FVIII two tothree times a week. Thus this treatment is rather complicated for anoutpatient population, especially in small children.

Thus currently the aim of many manufacturers of FVIII is to develop anext generation product with enhanced pharmacodynamic andpharmacokinetic properties, while maintaining all other productcharacteristics. Because improved polypeptide drugs with a longercirculation half-life would decrease the number of necessaryadministrations, chemical or enzymatic modification of the polypeptidedrugs is one of the preferred approaches to achieve this goal.

One such example is PEGylation of polypeptide drugs protecting andimproving their pharmacodynamic and pharmacokinetic profiles (Harris andChess, Nat Rev Drug Discov. 2003; 2:214-21). U.S. Pat. No. 6,037,452describes a poly(alkylene oxide)-FVIII or FIX conjugate, where theprotein is covalently bound to a poly(alkylene oxide) through carbonylgroups of said FVIII.

Even if these methods reduce inhibitor development they still would notabrogate the need for intravenous administration. The most elegantoption, making most of the drawbacks of hemophilia treatment discussedabove obsolete, would be the development of a low molecular weightcompound such as a peptide (peptidomimetic) with the capacity to improvecoagulation and which can be administered by a non-intravenous route.Though already discussed for many years (for example Kaufman and Pipe,Haemophilia 1998; 4.370-9; Llung, Thromb Haemost. 1999; 82:525-30) nosuch agent is currently available or in clinical development.

The current state of the art for the use of small peptides in bloodcoagulation is documented for example by the following publications:

D K Liles, D M Monroe and H R Roberts (1997) Blood Vol 90 No 10Supplement 1, 463a is a poster abstract disclosing a peptide 698-712from FVIII which can promote FIXa mediated activation of FX on aphospholipid surface. However, in the presence of FVIIIa, the peptideinhibits FIXa mediated activation of FX on a phospholipid surface. Todate, there has been no peer-reviewed publication by these authorsconfirming results disclosed in this poster abstract.

Blostein et al (2000) Biochemistry 39:12000-12006 discloses thatamphipathic alpha helices can interact with FIXa Gla domains andincreases activation of FX in the absence of phospholipid. The peptidesappeared to work independently of amino acid sequence by mimickingphospholipids. There is no suggestion to use such peptides in therapy.Under normal conditions, activated platelets provide the lipid[0]surface supporting coagulation. Since platelets are activated bythrombin, which is formed at sites of vascular injury, coagulationprocesses are restricted to the sites of injuries. It is highlyundesirable to provide the body with peptides that are generalsubstitutes for procoagulant lipids as this would cause systemiccoagulation and ultimately lead to disseminated intravascularcoagulation (DIC). Therefore, the peptides described by Blostein wouldnot be useful in therapy.

U.S. Pat. Nos. 7,109,170 and 6,624,289 disclose regions of the FIXaprotease domain that interact with FVIIIa. The peptides comprise theFVIIIa binding site of FIXa and inhibit binding of FIXa to FVIIIa.However, they are only useful as anticoagulants for preventing ortreating thrombosis.

US20010014456A1 discloses binding molecules for human FVIII andFVIII-like proteins. These polypeptides bind FVIII and/or FVIII-likepolypeptides and are useful for the detection and purification of humanFVIII and/or FVIII-like polypeptides from solutions such as blood orconditioned media.

In U.S. Pat. No. 7,033,590 FIX/FIXa activating antibodies and antibodyderivatives are used for increasing the amidolytic activity of FIXa, andfor treating blood coagulation disorders such as hemophilia A andhemorrhagic diathesis.

In U.S. Pat. No. 7,084,109 FVIIa antagonists are disclosed. Theseantagonists are peptides that inhibit FVIIa activity and are said to beuseful for prevention of arterial thrombosis in combination withthrombolytic therapy.

The listing or discussion of a prior-published document in thisspecification should not necessarily be taken as an acknowledgement thatthe document is part of the state of the art or is common generalknowledge.

There remains a great need in the art for a low molecular weight peptidewith procoagulant activity for treatment of patients with hemophilia A(FVIII deficiency). The present invention provides novel low molecularweight peptides with procoagulant activity which can be used for thenon-intravenous treatment of hemophilia A. The present prevention alsoprovides these novel peptides for the treatment of a deficiency in FV,FVII, FX and/or FXI.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a peptide or peptide derivativecomprising:

(i) WDLYFEIVW (SEQ ID NO: 1); or

(ii) a variant amino acid sequence comprising one, two, three or fourL-amino acid substitutions in WDLYFEIVW (SEQ ID NO: 1); or

(iii) the retro-inverso variant of the peptide or peptide derivative ofeither one of parts (i) and (ii),

wherein said peptide or peptide derivative has procoagulant activity.

For the avoidance of doubt, the sequence WDLYFEIVW (SEQ ID NO: 1) may berepresented as the L-amino acids Trp-Asp-Leu-Tyr-Phe-Glu-Ile-Val-Trpusing the three letter code for amino acids. The retro-inverso variantof WDLYFEIVW (SEQ ID NO: 1) is wviefyldw and comprises D-amino acids.

A second aspect of the invention provides a peptide or peptidederivative comprising:

-   -   (i) an amino acid sequence comprising imfwydcye; or    -   (ii) a variant amino acid sequence comprising one, two, three,        four, five or six amino acid substitutions in imfwydcye,

wherein said peptide or peptide derivative has procoagulant activity.

For the avoidance of doubt, the sequence cimfwydcye may be representedas D-amino acids ile-met-phe-trp-tyr-asp-cys-tyr-glu using the threeletter code for amino acids.

A third aspect of the invention provides a dual peptide comprising apeptide or peptide derivative of the first or second aspects of theinvention conjugated to a further peptide or peptide derivative of thefirst or second aspects of the invention, wherein the twopeptides/derivatives may be the same as or different from each other andwherein the dual peptide has procoagulant activity.

A fourth aspect of the invention provides a pharmaceutical compositioncomprising the peptide or peptide derivative of the first or secondaspects of the invention or the dual peptide of the third aspect of theinvention.

A fifth aspect of the invention provides a peptide or peptide derivativeof the first or second aspects or a dual peptide of the third aspect ofthe invention for treating a patient having a deficiency in FV, FVII,FVIII, FX and/or FXI.

A sixth aspect of the invention provides a use of a peptide or peptidederivative of the first or second aspects or a dual peptide of the thirdaspect of the invention in the manufacture of a medicament for thetreatment of a deficiency in FV, FVII, FVIII, FX and/or FXI in apatient.

A seventh aspect of the invention provides a method of treating apatient having a deficiency in FV, FVII, FVIII, FX and/or FXI comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition of the fourth aspect.

An eighth aspect of the invention provides a peptide or peptidederivative which has procoagulant activity, wherein the peptide orpeptide derivative is not FVIII or a fragment thereof and, wherein theprocoagulant activity is a thrombin generation time of 25, 50 or 100 μMof peptide, peptide derivative or dual peptide equivalent to that of atleast 100 mU/mL Factor Eight Inhibitor Bypassing Activity (FEIBA),preferably at least 300 mU/mL FEIBA, more preferably at least 900 mU/mLFEIBA, most preferably at least 1200 mU/mL FEIBA in the DefinedIntrinsic Thrombin Generation Assay.

A ninth aspect of the invention provides a peptide or peptide derivativewhich has procoagulant activity, wherein the peptide or peptidederivative is not FVIII or a fragment thereof and, wherein theprocoagulant activity is a thrombin generation time of 25, 50 or 100 μMof peptide, peptide derivative or dual peptide in a Defined IntrinsicThrombin Generation Assay peaking within 30 minutes, preferably within15 minutes and most preferably within 10 minutes.

A tenth aspect of the invention provides a peptide or peptide derivativewhich has procoagulant activity, wherein the peptide or peptidederivative is not FVIII or a fragment thereof and, wherein the peptideor peptide derivative can at least partially compensate for the absenceof biologically active FVIII when administered in an animal model ofsevere human hemophilia A.

DESCRIPTION OF FIGURES

FIG. 1: Effect of therapeutics approved for treatment of hemophilia onpeak thrombin generation and thrombin peak time in a definedDual-pathway thrombin generation assay

FIG. 2: Effect of A01 on FVIII −/− mouse bleeding model—blood loss

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The term “amino acid” within the scope of the present invention isintended to include all naturally occurring L α-amino acids. The one andthree letter abbreviations for naturally occurring amino acids are usedherein (Lehninger, Biochemistry, 2d ed., Worth Publishers, New York,1995: 71-92). The term “amino acid” also includes stereoisomers (forexample D-amino acids) and modifications of naturally occurring aminoacids, non-proteinogenic amino acids, and structures designed to mimicamino acids.

Modified and non-proteinogenic amino acids are described generally inGrant, Synthetic Peptides: A User's Guide, Oxford University Press,1992.

It is possible to provide, for example, improved stability andsolubility, resistance to protease degradation, and activity of thepeptide by the introduction of various amino acids that do not naturallyoccur, or by modification of the amino acid as discussed herein.

Non-proteinogenic amino acids may include but are not limited toβ-alanine (β-Ala), norvaline (Nva), norleucine (Nle), 4-aminobutyricacid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid(ε-Ahx), ornithine (orn), hydroxyproline (Hyp), sarcosine, citrulline,cysteic acid (Coh), and cyclohexylalanine, methioninesulfoxide (Meo),methioninesulfone (Moo), homoserinemethylester (Hsm), propargylglycine(Eag), 5-fluorotryptophan (5Fw), 6-fluorotryptophan (6Fw),3′,4′-dimethoxyphenyl-alanine (Ear), 3′,4′-difluorophenylalanine (Dff),4′-fluorophenyl-alanine (Pff), 1-naphthyl-alanine (1Ni),1-methyltryptophan (1Mw), penicillamine (Pen), homoserine (HSe).Further, such amino acids may include but are not limited to, α-aminoisobutyric acid, t-butylglycine, t-butylalanine, phenylglycine (Phg),benzothienylalanine (Bta), L-homo-cysteine (L-Hcys),N-methyl-phenylalanine (NMF), 2-thienylalanine (Thi),3,3-diphenylalanine (Ebw), homophenylalanine (Hfe), s-benzyl-L-cysteine(Ece) or cyclohexylalanine (Cha). These and other non-proteinogenicamino acids may exist as D- or L-isomers. Where no indication of theisomer is given, the L-isomer is intended.

Structures which are designed to mimic amino acids are compounds inwhich the amino and/or carboxyl group of an amino acid is replaced byanother group. Non-limiting examples are the incorporation ofthioamides, ureas, thioureas, acylhydrazides, esters, olefines,sulfonamides, phosphoric acid amides, ketones, alcohols, boronic acidamides, benzodiazepines and other aromatic or non-aromatic heterocycles(for a review see M. A. Estiarte, D. H. Rich in Burgers MedicinalChemistry, 6th edition, volume 1, part 4, John Wiley & Sons, New York,2002). If these structures are included in a peptide derivative they areusually connected to the rest of the peptide derivative with at leastone of the above mentioned functional groups instead of an amide bond.

By “peptide” we include not only molecules in which amino acid residuesare joined by peptide (—CO—NH—) linkages but also molecules in which thepeptide bond is reversed. A “retro modified” peptide is a peptide thatis made up of amino acids in which the amino acid residues are assembledin opposite direction to the native peptide with respect to which it isretro modified. Where the native peptide comprises L-amino acids, the“retro modified” peptide will also comprise L-amino acids. However,where the native peptide comprises D-amino acids, the “retro modified”peptide will comprise D-amino acids. Retro peptides contain NH—CO bondsinstead of CO—NH peptide bonds. An “inverso modified” peptide is apeptide in which the amino acid residues are assembled in the samedirection as the native peptide with respect to which it is inversomodified, but the chirality of the amino acids is inverted. Thus, wherethe native peptide comprises L-amino acids, the “inverso modified”peptide will comprise D-amino acids. Where the native peptide comprisesD-amino acids, the “inverso modified” peptide will comprise L-aminoacids. Inverso peptides still have CO—NH peptide bonds. A “retro-inversomodified” peptide refers to a peptide that is made up of amino acidresidues which are assembled in the opposite direction and which haveinverted chirality with respect to the native peptide to which it isretro-inverso modified. A retro-inverso analogue has reversed terminiand reversed direction of peptide bonds (i.e. NH—CO) while approximatelymaintaining the topology of the side chains as in the native peptidesequence. Guichard et al (1994) Proc. Natl. Acad. Sci USA 91:9765-9769described that a retro-inverso peptide mimicked the structure andantigenic activity of the natural L-peptide IRGERA, but not of the D-and retro peptides. Such retro-inverso peptidomimetics may be made usingmethods known in the art, for example such as those described in Meziereet al (1997) J. Immunol. 159, 3230-3237, incorporated herein byreference. Partial retro-inverso peptide analogues are polypeptides inwhich only part of the sequence is reversed and replaced withenantiomeric amino acid residues. Processes for making such analoguesare described in Pessi, A., Pinori, M., Verdini, A. S. & Viscomi, G. C.(1987) “Totally solid phase synthesis of peptide(s)-containingretro-inverted peptide bond, using crosslinked sarcosinyl copolymer assupport”, European Patent 97994-B.

Conventionally, L-amino acids are designated using upper case, andD-amino acids are designated in lower case. The peptides and peptidederivatives of the invention are designated in their preferred form, butwithout limiting them to the preferred form. The peptide of the firstaspect of the invention is designated as comprising WDLYFEIVW (SEQ IDNO: 1) or a variant thereof. The peptide of the first aspect of theinvention may also be the retro-inverso variant of WDLYFEIVW (SEQ IDNO: 1) or a variant thereof, namely wviefyldw or a variant thereof. Thepeptide of the second aspect of the invention is designated ascomprising cimfwydcye or a variant thereof.

Conventionally, where the amino acids are joined by peptide bonds, apeptide is represented such that the amino group at the N-terminusappears to the left and the carboxyl group at the C-terminus to theright. Peptides and peptide derivatives according to the presentinvention are represented in this manner.

A “peptide derivative” contains a modification of one or more amino acidresidues or a linker group or other covalently linked group.

Examples of derivatives include N-acyl derivatives of the amino terminalor of another free amino group, esters of the carboxyl terminal or ofanother free carboxyl or hydroxy group, amides of the carboxyl terminalor of another free carboxyl group produced by reaction with ammonia orwith a suitable amine, glycosylated derivatives, hydroxylatedderivatives, nucleotidylated derivatives, ADP-ribosylated derivatives,pegylated derivatives, phosphorylated derivatives, derivativesconjugated to lipophilic moieties, and derivatives conjugated to anantibody or other biological ligand. Also included among the chemicalderivatives are those obtained by modification of the peptide bond—CO—NH—, for example by reduction to —CH₂—NH— or alkylation to—CO—N(alkyl)-.

A preferred derivatisation is C-terminal amidation. C-terminal amidationof a peptide removes the negative charge of the C terminus. Peptidederivatives having a C-terminal amide are represented with “NH₂” at theC-terminus, for example Ac-WDLYFEIVW-NH₂ (SEQ ID NO: 1). Anotherpreferred derivatisation is N-terminal acetylation. This removes thepositive charge at the N-terminus. Blocking of the C- or N-terminus,such as by C-terminal amidation or N-terminal acetylation, may improveproteolytic stability due to reduced susceptibility to exoproteolyticdigestion.

Suitable linkers include the flexible linker4,7,10-trioxa-1,13-tridecanediamine (Ttds), glycine, 6-aminohexanoicacid, beta-alanine, or combinations of Ttds, glycine, 6-aminohexanoicacid and beta-alanine.

The peptides of this invention can be produced by chemical synthesis,recombinant DNA technology, biochemical or enzymatic fragmentation oflarger molecules, combinations of the foregoing or by any other method.

Peptides (at least those containing peptide linkages between amino acidresidues) may be synthesised by the Fmoc strategy of solid-phase peptidesynthesis as described in “Fmoc Solid Phase Peptide Synthesis—APractical Approach”, edited by W. C. Chan, P. D. White, OxfordUniversity Press, New York 2000 and references therein. TemporaryN-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl(Fmoc) group. Repetitive cleavage of this highly base-labile protectinggroup is effected using 20% piperidine in N,N-dimethylformamide.Side-chain functionalities may be protected as their butyl ethers (inthe case of serine, threonine and tyrosine), butyl esters (in the caseof glutamic acid and aspartic acid), butyloxycarbonyl derivative (in thecase of lysine and histidine), trityl derivative (in the case ofcysteine, asparagine and glutamine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). The solid-phase support is based on a polydimethyl-acrylamidepolymer constituted from the three monomers dimethylacrylamide(backbone-monomer), bisacryloylethylene diamine (cross linker) andacryloylsarcosine methyl ester (functionalising agent). Thepeptide-to-resin cleavable linked agent used is the acid-labile4-hydroxymethyl-phenoxyacetic acid derivative, or in case of C-terminalamides, the Rink-amide linker. All amino acid derivatives are added astheir preformed symmetrical anhydride derivatives with the exception ofasparagine and glutamine, which are added using a reversedN,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated couplingprocedure. All coupling and deprotection reactions are monitored usingninhydrin, trinitrobenzene sulphonic acid or isotin test procedures.Upon completion of synthesis, peptides are cleaved from the resinsupport with concomitant removal of side-chain protecting groups bytreatment with 95% trifluoroacetic acid containing a 50% scavenger mix.Scavengers commonly used are ethanedithiol, phenol, anisole and water,the exact choice depending on the constituent amino acids of the peptidebeing synthesised. Trifluoroacetic acid is removed by evaporation invacuo, with subsequent trituration with diethyl ether affording thecrude peptide. Any scavengers present are removed by a simple extractionprocedure which on lyophilisation of the aqueous phase affords the crudepeptide free of scavengers. Reagents for peptide synthesis are generallyavailable from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.Purification may be effected by any one, or a combination of, techniquessuch as size exclusion chromatography, ion-exchange chromatography,affinity chromatography, differential solubility and (principally)reverse-phase high performance liquid chromatography. Analysis ofpeptides may be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, amino-acidanalysis after acid hydrolysis and by fast atom bombardment (FAB) massspectrometric analysis.

SPOT-synthesis, which allows the positional addressable, chemicalsynthesis of peptides on continuous cellulose membranes may be also used(R Frank Tetrahedron (1992) 48, 9217).

As an alternative to solid phase peptide synthesis techniques, peptidesmay also be produced by recombinant protein expression or in vitrotranslation systems (Sambrook et al, “Molecular cloning: A laboratorymanual”, 2001, 3^(rd) edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). Of course, it is only peptides which containnaturally occurring amino acid residues joined by naturally-occurringpeptide bonds which are encodable by a polynucleotide. Such methods arepreferred over solid phase peptide synthesis techniques where thepeptide is particularly large, such as larger than 50 amino acids, orlarger than 100 amino acids.

A “variant” amino acid sequence as defined in relation to the firstaspect of the invention may comprise one, two, three or four L-aminoacid substitutions in WDLYFEIVW (SEQ ID NO: 1).

Preferably, the variant amino acid sequence comprises an amino acidsequence comprising X₁X₂X₃YX₄EX₅X₆X₇ wherein X₁ is W, L or P, X₂ is D orS, X₃ is L or F, X₄ is F, Phg, L, Ebw, Pff, Thi, 1Ni, Hfe, Ece or Cha,X₅ is I or F, X₆ is S, V or G and X₇ is W or L (SEQ ID NO: 1).

More preferably, the variant amino acid sequence comprises an amino acidsequence comprising X₁X₂X₃YX₄EX₅X₆X₇ wherein X₁ is W or L, X₂ is D or S,X₃ is L or F, X₄ is F, Phg or L, X₅ is I or F, X₆ is S, V or G and X₇ isW or L (SEQ ID NO: 1).

A “variant” amino acid sequence as defined in relation to the secondaspect of the invention may comprise one, two, three, four, five or sixamino acid substitutions in imfwydcye.

Preferably, at least one, two, three, four, five or six of saidsubstitutions in imfwydcye are D-amino acids.

Any substitution within the variant may be non-conservative orconservative.

By “conservative substitutions” we mean substitutions within thefollowing groups: Val, Ile, Leu, Ala, Met; Asp, Glu; Asn, Gln; Ser, Thr,Gly, Ala; Lys, Arg, His; and Phe, Tyr, Trp.

Preferably, the peptide or peptide derivative of the first aspect of theinvention comprises RMEFDVWDLYFEIVW (SEQ ID NO: 2) or RMKFDVWDLYFEIVW(SEQ ID NO: 2); or a variant amino acid sequence comprising one, two,three, four, five or six amino acid substitutions in RMEFDVWDLYFEIVW(SEQ ID NO: 2) or RMKFDVWDLYFEIVW (SEQ ID NO: 2).

For the avoidance of doubt, the sequence RMEFDVWDLYFEIVW (SEQ ID NO: 2)may be represented asArg-Met-Glu-Phe-Asp-Val-Trp-Asp-Leu-Tyr-Phe-Glu-Ile-Val-Trp using thethree letter code for amino acids. RMKFDVWDLYFEIVW (SEQ ID NO: 2) may berepresented asArg-Met-Lys-Phe-Asp-Val-Trp-Asp-Leu-Tyr-Phe-Glu-Ile-Val-Trp using thethree letter code for amino acids.

More preferably, the variant amino acid sequence comprises an amino acidsequence comprising X₈X₉X₁₀FDVX₁X₂X₃YX₄EX₅X₆X₇ wherein X₈ is R or P, X₉is M, Nva, Moo, N, Nle, Meo, Q, Eag, X₁₀ is E, K or D, X₁ is W, L or P,X₂ is D or S, X₃ is L or F, X₄ is F, Phg, L, Ebw, Pff, Thi, 1Ni, Hfe,Ece, Cha, X₅ is I or F, X₆ is S, V or G and X₇ is W or L (SEQ ID NO: 2).

More preferably, the variant amino acid sequence comprises an amino acidsequence comprising X₈X₉X₁₀FDVX₁X₂X₃YX₄EX₅X₆X₇ wherein X₈ is R or P, X₉is M or Nva, X₁₀ is E, K or D, X₁ is W or L, X₂ is D or S, X₃ is L or F,X₄ is F, Phg or L, X₅ is I or F, X₆ is S, V or G and X₇ is W or L (SEQID NO: 2).

Suitably, the peptide or peptide derivative of the first aspect of theinvention is a peptide or peptide derivative as represented in the tablebelow, or comprises or consists of the amino acid sequence of a peptideor peptide derivative as represented in tables 1 to 3 below:

TABLE 1 Most preferred peptides Peptide Sequence SEQ ID NO: 2 A01Ac-RMKFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A02 Ac-PMKFDVWDLYFEIVW-NH₂SEQ ID NO: 2 A03 Ac-RMDFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A04Ac-RMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 1 A05 Ac-WDLYFEIVW-NH₂ SEQ ID NO: 3A06 Ac-WDLYFEIVWE SEQ ID NO: 1 A07 Ac-WDLYFEIVW-ttds-E SEQ ID NO: 2 A08ttds-RMEFDVWDLYFEIVW- ttds-NH₂ SEQ ID NO: 4 A09 ERMEFDVWDLYFEIVW-NH₂SEQ ID NO: 5 A12 ERXEFDVWDLYFEIVW-NH₂ X is Nva A13 ttds-RMEFDVWDLYXEIVW-ttds-NH₂ X is Phg SEQ ID NO: 6 A14 Ac-WSLYFEIVWE SEQ ID NO: 1 A15Ac-WDLYFEISW-ttds-E SEQ ID NO: 2 A16 PEG5000-RMKFDVWDLYFEIVW-NH₂SEQ ID NO: 6 A17 PEG5000-WSLYFEIVWE SEQ ID NO: 4 A18PEG5000-ERMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 7 A19 Ac-VWDLYFEIVW-NH₂SEQ ID NO: 8 A21 Ac-FDVWDLYFEIVW-NH₂ SEQ ID NO: 9 A24 EWDLYFEIVW-NH₂SEQ ID NO: 1 A25 E-ttds-WDLYFEIVW-NH₂ SEQ ID NO: 1 A26Ac-WDLYFEIVW-ttds-E-NH₂ SEQ ID NO: 2 A27 Ac-RMEFDVWDLYFEIVW SEQ ID NO: 2A28 RMEFDVWDLYFEIVW SEQ ID NO: 2 A29 Ac-K-ttds-RMEFDVWDLYFEIVW- NH₂SEQ ID NO: 10 A30 Ac-RMEFDVWDLYFEIVWK SEQ ID NO: 10 A31Ac-RMEFDVWDLYFEIVWK-NH₂ SEQ ID NO: 2 A32 Ac-RMEFDVWDLYFEIVW-ttds-K- NH₂SEQ ID NO: 11 A33 Ac-WDLYFEISWE SEQ ID NO: 12 A34 Ac-WDLYLEIVWESEQ ID NO: 13 A35 Ac-WDLYFEIVLE SEQ ID NO: 1 A38 WDLYFEIVW SEQ ID NO: 2A49 RMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A50 Ac-RMEFDVWDLYFEIVW-ttds-NH₂SEQ ID NO: 14 A52 Ac-KRMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A53K-ttds-RMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A54 Ac-RMEFDVWDLYFEIVW-ttds-KSEQ ID NO: 1 A55 Ac-LDLYFEIVW-ttds-E SEQ ID NO: 1 A56Ac-WDLYFEIVL-ttds-E SEQ ID NO: 15 A57 E-RMEFDVLDLYFEIVW-NH₂SEQ ID NO: 16 A58 E-RMEFDVWDLYFEIVL-NH₂ SEQ ID NO: 17 A84 Ac-WDFYFEIVWESEQ ID NO: 18 A85 Ac-WDLYFEFVWE SEQ ID NO: 19 A86 Ac-LDLYFEIVWESEQ ID NO: 20 A87 Ac-WDLYFEIGWE SEQ ID NO: 21 A89 Ac-WDLYLEISLE A90Ac-WDLYXEIVLE X is Phg A91 Ac-WSLYXEIVWE X is Phg SEQ ID NO: 22 A92Ac-LDLYFEIVLE SEQ ID NO: 23 A93 Ac-LDLYFEISLE A94 Ac-LDLYXEISWE X is PhgSEQ ID NO: 24 A95 Ac-LSLYFEIVWE SEQ ID NO: 25 A96 Ac-LSLYFEIVLESEQ ID NO: 26 A97 Ac-LSLYFEISLE

TABLE 2 Preferred peptides Peptide Sequence SEQ ID NO: 1 A20Ac-WDLYFEIVW-ttds-K SEQ ID NO: 27 A22 Ac-DVWDLYFEIVW-NH₂ A23Ac-wviefyldwvdfkmr-NH₂ SEQ ID NO: 1 A37 Ac-WDLYFEIVW SEQ ID NO: 1 A39Ac-ttds-WDLYFEIVW-NH₂ SEQ ID NO: 1 A40 ttds-WDLYFEIVW-NH₂ SEQ ID NO: 1A41 Ac-WDLYFEIVW-ttds-NH₂ SEQ ID NO: 1 A42 Ac-ttds-WDLYFEIVW-ttds-NH₂SEQ ID NO: 1 A43 ttds-WDLYFEIVW-ttds SEQ ID NO: 1 A44ttds-WDLYFEIVW-ttds-NH₂ SEQ ID NO: 28 A45 Ac-KWDLYFEIVW-NH₂ SEQ ID NO: 1A46 Ac-K-ttds-WDLYFEIVW-NH₂ SEQ ID NO: 29 A47 Ac-WDLYFEIVWKSEQ ID NO: 29 A48 Ac-WDLYFEIVWK-NH₂ A71 E-R(Moo)EFDVWDLYFEIVW-NH₂SEQ ID NO: 30 A73 E-RNEFDVWDLYFEIVW-NH₂ A78 ttds-RMEFDVWDLY(Ebw)EIVW-ttds-NH₂ A83 ttds-RMEFDVWDLY(Pff)EIVW- ttds-NH₂ SEQ ID NO: 31 A88Ac-PDLYFEIVWE SEQ ID NO: 32 A98 Ac-LSLYLEIVLE SEQ ID NO: 33 A99Ac-LSLYLEISLE A100 Ac-LSLYXEIVLE X is Phg SEQ ID NO: 1 A101Ac-WDLYFEIVW-ttds-K-NH₂

TABLE 3 Active peptides Peptide Sequence SEQ ID NO: 34 A10E-PMKFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A11 ttds-RMDFDVWDLYFEIVW- ttds-NH₂SEQ ID NO: 2 A16 PEG5000-RMKFDVWDLYFEIVW-NH₂ SEQ ID NO: 1 A36WDLYFEIVW-NH₂ SEQ ID NO: 14 A51 KRMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A59ttds-PMKFDVWDLYFEIVW- ttds-NH₂ SEQ ID NO: 35 A60 E-RMDFDVWDLYFEIVW-NH₂SEQ ID NO: 2 A61 (Coh)-ttds-RMEFDVWDLYFEIVW- ttds-NH₂ SEQ ID NO: 2 A62Glucosyl-aminooxyacetyl- ttds-RMEFDVWDLYFEIVW- ttds-NH₂ A63Ac-P(Moo)KFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A64 Ac-P(Nle)KFDVWDLYFEIVW-NH₂SEQ ID NO: 2 A65 Ac-PNKFDVWDLYFEIVW-NH₂ A66 Ac-R(Moo)DFDVWDLYFEIVW-NH₂SEQ ID NO: 2 A67 Ac-R(Nle)DFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A68Ac-RNDFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A69 ttds-R(Nle)EFDVWDLYFEIVW-ttds-NH₂ SEQ ID NO: 2 A70 ttds-RNEFDVWDLYFEIVW- ttds-NH₂ SEQ ID NO: 321A72 E-R(Nle)EFDVWDLYFEIVW-NH₂ A74 E-R(Meo)EFDVWDLYFEIVW-NH₂SEQ ID NO: 36 A75 E-R(Gln)EFDVWDLYFEIVW-NH₂ A76E-R(Eag)EFDVWDLYFEIVW-NH₂ A77 ttds-RMEFDVWDLY(Thi)EIVW- ttds-NH₂ A79ttds-RMEFDVWDLY(1Ni)EIVW- ttds-NH₂ A80 ttds-RMEFDVWDLY(Hfe)EIVW-ttds-NH₂ A81 ttds-RMEFDVWDLY(Ece)EIVW- ttds-NH₂ A82ttds-RMEFDVWDLY(Cha)EIVW- ttds-NH₂ SEQ ID NO: 28 A102 KWDLYFEIVW-NH₂SEQ ID NO: 1 A103 K-ttds-WDLYFEIVW-NH₂

In the above tables, -ttds- is 4,7,10-trioxa-1,13-tridecanediamine. “N”is asparagine. “NH₂” is a C-terminal amide group.

Preferably, the peptide or peptide derivative of the first aspect of theinvention does not comprise or consist of a peptide represented in thelist below:

(SEQ ID NO: 37) AMKFDVWDLYFEIVW, (SEQ ID NO: 38) CMKFDVWDLYFEIVW,(SEQ ID NO: 39) DMKFDVWDLYFEIVW, (SEQ ID NO: 40) EMKFDVWDLYFEIVW,(SEQ ID NO: 41) FMKFDVWDLYFEIVW, (SEQ ID NO: 42) GMKFDVWDLYFEIVW,(SEQ ID NO: 43) HMKFDVWDLYFEIVW, (SEQ ID NO: 44) IMKFDVWDLYFEIVW,(SEQ ID NO: 45) KMKFDVWDLYFEIVW, (SEQ ID NO: 46) LMKFDVWDLYFEIVW,(SEQ ID NO: 47) MMKFDVWDLYFEIVW, (SEQ ID NO: 48) NMKFDVWDLYFEIVW,(SEQ ID NO: 49) QMKFDVWDLYFEIVW, (SEQ ID NO: 50) SMKFDVWDLYFEIVW,(SEQ ID NO: 51) TMKFDVWDLYFEIVW, (SEQ ID NO: 52) VMKFDVWDLYFEIVW,(SEQ ID NO: 53) WMKFDVWDLYFEIVW, (SEQ ID NO: 54) YMKFDVWDLYFEIVW,(SEQ ID NO: 55) RAKFDVWDLYFEIVW, (SEQ ID NO: 56) RCKFDVWDLYFEIVW,(SEQ ID NO: 57) RDKFDVWDLYFEIVW, (SEQ ID NO: 58) REKFDVWDLYFEIVW,(SEQ ID NO: 59) RFKFDVWDLYFEIVW, (SEQ ID NO: 60) RGKFDVWDLYFEIVW,(SEQ ID NO: 61) RHKFDVWDLYFEIVW, (SEQ ID NO: 62) RIKFDVWDLYFEIVW,(SEQ ID NO: 63) RKKFDVWDLYFEIVW, (SEQ ID NO: 64) RLKFDVWDLYFEIVW,(SEQ ID NO: 65) RNKFDVWDLYFEIVW, (SEQ ID NO: 66) RPKFDVWDLYFEIVW,(SEQ ID NO: 67) RQKFDVWDLYFEIVW, (SEQ ID NO: 68) RRKFDVWDLYFEIVW,(SEQ ID NO: 69) RSKFDVWDLYFEIVW, (SEQ ID NO: 70) RTKFDVWDLYFEIVW,(SEQ ID NO: 71) RVKFDVWDLYFEIVW, (SEQ ID NO: 72) RWKFDVWDLYFEIVW,(SEQ ID NO: 73) RYKFDVWDLYFEIVW, (SEQ ID NO: 74) RMAFDVWDLYFEIVW,(SEQ ID NO: 75) RMCFDVWDLYFEIVW, (SEQ ID NO: 76) RMFFDVWDLYFEIVW,(SEQ ID NO: 77) RMGFDVWDLYFEIVW, (SEQ ID NO: 78) RMHFDVWDLYFEIVW,(SEQ ID NO: 79) RMIFDVWDLYFEIVW, (SEQ ID NO: 80) RMLFDVWDLYFEIWV,(SEQ ID NO: 81) RMMFDVWDLYFEIVW, (SEQ ID NO: 82) RMNFDVWDLYFEIVW,(SEQ ID NO: 83) RMPFDVWDLYFEIVW, (SEQ ID NO: 84) RMQFDVWDLYFEIVW,(SEQ ID NO: 85) RMRFDVWDLYFEIVW, (SEQ ID NO: 86) RMSFDVWDLYFEIVW,(SEQ ID NO: 87) RMTFDVWDLYFEIVW, (SEQ ID NO: 88) RMVFDVWDLYFEIVW,(SEQ ID NO: 89) RMWFDVWDLYFEIVW, (SEQ ID NO: 90) RMYFDVWDLYFEIVW,(SEQ ID NO: 91) RMKADVWDLYFEIVW, (SEQ ID NO: 92) RMKCDVWDLYFEIVW,(SEQ ID NO: 93) RMKDDVWDLYFEIVW, (SEQ ID NO: 94) RMKEDVWDLYFEIVW,(SEQ ID NO: 95) RMKGDVWDLYFEIVW, (SEQ ID NO: 96) RMKHDVWDLYFEIVW,(SEQ ID NO: 97) RMKIDVWDLYFEIVW, (SEQ ID NO: 98) RMKKDVWDLYFEIVW,(SEQ ID NO: 99) RMKLDVWDLYFEIVW, (SEQ ID NO: 100) RMKMDVWDLYFEIVW,(SEQ ID NO: 101) RMKNDVWDLYFEIVW, (SEQ ID NO: 102) RMKPDVWDLYFEIVW,(SEQ ID NO: 103) RMKQDVWDLYFEIVW, (SEQ ID NO: 104) RMKRDVWDLYFEIVW,(SEQ ID NO: 105) RMKSDVWDLYFEIVW, (SEQ ID NO: 106) RMKTDVWDLYFEIVW,(SEQ ID NO: 107) RMKVDVWDLYFEIVW, (SEQ ID NO: 108) RMKWDVWDLYFEIVW,(SEQ ID NO: 109) RMKYDVWDLYFEIVW, (SEQ ID NO: 110) RMKFAVWDLYFEIVW,(SEQ ID NO: 111) RMKFCVWDLYFEIVW, (SEQ ID NO: 112) RMKFEVWDLYFEIVW,(SEQ ID NO: 113) RMKFFVWDLYFEIVW, (SEQ ID NO: 114) RMKFGVWDLYFEIVW,(SEQ ID NO: 115) RMKFHVWDLYFEIVW, (SEQ ID NO: 116) RMKFIVWDLYFEIVW,(SEQ ID NO: 117) RMKFKVWDLYFEIVW, (SEQ ID NO: 118) RMKFLVWDLYFEIVW,(SEQ ID NO: 119) RMKFMVWDLYFEIVW, (SEQ ID NO: 120) RMKFNVWDLYFEIVW,(SEQ ID NO: 121) RMKFPVWDLYFEIVW, (SEQ ID NO: 122) RMKFQVWDLYFEIVW,(SEQ ID NO: 123) RMKFRVWDLYFEIVW, (SEQ ID NO: 124) RMKFSVWDLYFEIVW,(SEQ ID NO: 125) RMKFTVWDLYFEIVW, (SEQ ID NO: 126) RMKFVVWDLYFEIVW,(SEQ ID NO: 127) RMKFWVWDLYFEIVW, (SEQ ID NO: 128) RMKFYVWDLYFEIVW,(SEQ ID NO: 129) RMKFDAWDLYFEIVW, (SEQ ID NO: 130) RMKFDCWDLYFEIVW,(SEQ ID NO: 131) RMKFDDWDLYFEIVW, (SEQ ID NO: 132) RMKFDEWDLYFEIVW,(SEQ ID NO: 133) RMKFDFWDLYFEIVW, (SEQ ID NO: 134) RMKFDGWDLYFEIVW,(SEQ ID NO: 135) RMKFDHWDLYFEIVW, (SEQ ID NO: 136) RMKFDIWDLYFEIVW,(SEQ ID NO: 137) RMKFDKWDLYFEIVW, (SEQ ID NO: 138) RMKFDLWDLYFEIVW,(SEQ ID NO: 139) RMKFDMWDLYFEIWV, (SEQ ID NO: 140) RMKFDNWDLYFEIVW,(SEQ ID NO: 141) RMKFDPWDLYFEIVW, (SEQ ID NO: 142) RMKFDQWDLYFEIVW,(SEQ ID NO: 143) RMKFDRWDLYFEIVW, (SEQ ID NO: 144) RMKFDSWDLYFEIVW,(SEQ ID NO: 145) RMKFDTWDLYFEIVW, (SEQ ID NO: 146) RMKFDWWDLYFEIVW,(SEQ ID NO: 147) RMKFDYWDLYFEIVW, (SEQ ID NO: 148) RMKFDVADLYFEIVW,(SEQ ID NO: 149) RMKFDVCDLYFEIVW, (SEQ ID NO: 150) RMKFDVDDLYFEIVW,(SEQ ID NO: 151) RMKFDVEDLYFEIVW, (SEQ ID NO: 152) RMKFDVFDLYFEIVW,(SEQ ID NO: 153) RMKFDVGDLYFEIVW, (SEQ ID NO: 154) RMKFDVHDLYFEIVW,(SEQ ID NO: 155) RMKFDVIDLYFEIVW, (SEQ ID NO: 156) RMKFDVKDLYFEIVW,(SEQ ID NO: 157) RMKFDVLDLYFEIVW, (SEQ ID NO: 158) RMKFDVMDLYFEIVW,(SEQ ID NO: 159) RMKFDVNDLYFEIVW, (SEQ ID NO: 160) RMKFDVPDLYFEIVW,(SEQ ID NO: 161) RMKFDVQDLYFEIVW, (SEQ ID NO: 162) RMKFDVRDLYFEIVW,(SEQ ID NO: 163) RMKFDVSDLYFEIVW, (SEQ ID NO: 164) RMKFDVTDLYFEIVW,(SEQ ID NO: 165) RMKFDVVDLYFEIVW, (SEQ ID NO: 166) RMKFDVYDLYFEIVW,(SEQ ID NO: 167) RMKFDVWALYFEIVW, (SEQ ID NO: 168) RMKFDVWCLYFEIVW,(SEQ ID NO: 169) RMKFDVWELYFEIVW, (SEQ ID NO: 170) RMKFDVWFLYFEIVW,(SEQ ID NO: 171) RMKFDVWGLYFEIVW, (SEQ ID NO: 172) RMKFDVWHLYFEIVW,(SEQ ID NO: 173) RMKFDVWILYFEIVW, (SEQ ID NO: 174) RMKFDVWKLYFEIVW,(SEQ ID NO: 175) RMKFDVWLLYFEIVW, (SEQ ID NO: 176) RMKFDVWMLYFEIVW,(SEQ ID NO: 177) RMKFDVWNLYFEIVW, (SEQ ID NO: 178) RMKFDVWPLYFEIVW,(SEQ ID NO: 179) RMKFDVWQLYFEIVW, (SEQ ID NO: 180) RMKFDVWRLYFEIVW,(SEQ ID NO: 181) RMKFDVWSLYFEIVW, (SEQ ID NO: 182) RMKFDVWTLYFEIVW,(SEQ ID NO: 183) RMKFDVWVLYFEIVW, (SEQ ID NO: 184) RMKFDVWWLYFEIVW,(SEQ ID NO: 185) RMKFDVWYLYFEIVW, (SEQ ID NO: 186) RMKFDVWDAYFEIVW,(SEQ ID NO: 187) RMKFDVWDCYFEIVW, (SEQ ID NO: 188) RMKFDVWDDYFEIVW,(SEQ ID NO: 189) RMKFDVWDEYFEIVW, (SEQ ID NO: 190) RMKFDVWDFYFEIVW,(SEQ ID NO: 191) RMKFDVWDGYFEIVW, (SEQ ID NO: 192) RMKFDVWDHYFEIVW,(SEQ ID NO: 193) RMKFDVWDIYFEIVW, (SEQ ID NO: 194) RMKFDVWDKYFEIVW,(SEQ ID NO: 195) RMKFDVWDMYFEIVW, (SEQ ID NO: 196) RMKFDVWDNYFEIVW,(SEQ ID NO: 197) RMKFDVWDPYFEIVW, (SEQ ID NO: 198) RMKFDVWDQYFEIVW,(SEQ ID NO: 199) RMKFDVWDRYFEIVW, (SEQ ID NO: 200) RMKFDVWDSYFEIVW,(SEQ ID NO: 201) RMKFDVWDTYFEIVW, (SEQ ID NO: 202) RMKFDVWDVYFEIVW,(SEQ ID NO: 203) RMKFDVWDWYFEIVW, (SEQ ID NO: 204) RMKFDVWDYYFEIVW,(SEQ ID NO: 205) RMKFDVWDLAFEIVW, (SEQ ID NO: 206) RMKFDVWDLCFEIVW,(SEQ ID NO: 207) RMKFDVWDLDFEIVW, (SEQ ID NO: 208) RMKFDVWDLEFEIVW,(SEQ ID NO: 209) RMKFDVWDLFFEIVW, (SEQ ID NO: 210) RMKFDVWDLGFEIVW,(SEQ ID NO: 211) RMKFDVWDLHFEIVW, (SEQ ID NO: 212) RMKFDVWDLIFEIVW,(SEQ ID NO: 213) RMKFDVWDLKFEIVW, (SEQ ID NO: 214) RMKFDVWDLLFEIVW,(SEQ ID NO: 215) RMKFDVWDLMFEIVW, (SEQ ID NO: 216) RMKFDVWDLNFEIVW,(SEQ ID NO: 217) RMKFDVWDLPFEIVW, (SEQ ID NO: 218) RMKFDVWDLQFEIVW,(SEQ ID NO: 219) RMKFDVWDLRFEIVW, (SEQ ID NO: 220) RMKFDVWDLSFEIVW,(SEQ ID NO: 221) RMKFDVWDLTFEIVW, (SEQ ID NO: 222) RMKFDVWDLVFEIVW,(SEQ ID NO: 223) RMKFDVWDLWFEIVW, (SEQ ID NO: 224) RMKFDVWDLYAEIVW,(SEQ ID NO: 225) RMKFDVWDLYCEIVW, (SEQ ID NO: 226) RMKFDVWDLYDEIVW,(SEQ ID NO: 227) RMKFDVWDLYEEIVW, (SEQ ID NO: 228) RMKFDVWDLYGEIVW,(SEQ ID NO: 229) RMKFDVWDLYHEIVW, (SEQ ID NO: 230) RMKFDVWDLYIEIVW,(SEQ ID NO: 231) RMKFDVWDLYKEIVW, (SEQ ID NO: 232) RMKFDVWDLYLEIVW,(SEQ ID NO: 233) RMKFDVWDLYMEIVW, (SEQ ID NO: 234) RMKFDVWDLYNEIVW,(SEQ ID NO: 235) RMKFDVWDLYPEIVW, (SEQ ID NO: 236) RMKFDVWDLYQEIVW,(SEQ ID NO: 237) RMKFDVWDLYREIVW, (SEQ ID NO: 238) RMKFDVWDLYSEIVW,(SEQ ID NO: 239) RMKFDVWDLYTEIVW, (SEQ ID NO: 240) RMKFDVWDLYVEIVW,(SEQ ID NO: 241) RMKFDVWDLYWEIVW, (SEQ ID NO: 242) RMKFDVWDLYYEIVW,(SEQ ID NO: 243) RMKFDVWDLYFAIVW, (SEQ ID NO: 244) RMKFDVWDLYFCIVW,(SEQ ID NO: 245) RMKFDVWDLYFDIVW, (SEQ ID NO: 246) RMKFDVWDLYFFIVW,(SEQ ID NO: 247) RMKFDVWDLYFGIVW, (SEQ ID NO: 248) RMKFDVWDLYFHIVW,(SEQ ID NO: 249) RMKFDVWDLYFIIVW, (SEQ ID NO: 250) RMKFDVWDLYFKIVW,(SEQ ID NO: 251) RMKFDVWDLYFLIVW, (SEQ ID NO: 252) RMKFDVWDLYFMIVW,(SEQ ID NO: 253) RMKFDVWDLYFNIVW, (SEQ ID NO: 254) RMKFDVWDLYFPIVW,(SEQ ID NO: 255) RMKFDVWDLYFQIVW, (SEQ ID NO: 256) RMKFDVWDLYFRIVW,(SEQ ID NO: 257) RMKFDVWDLYFSIVW, (SEQ ID NO: 258) RMKFDVWDLYFTIVW,(SEQ ID NO: 259) RMKFDVWDLYFVIVW, (SEQ ID NO: 260) RMKFDVWDLYFWIVW,(SEQ ID NO: 261) RMKFDVWDLYFYIVW, (SEQ ID NO: 262) RMKFDVWDLYFEAVW,(SEQ ID NO: 263) RMKFDVWDLYFECVW, (SEQ ID NO: 264) RMKFDVWDLYFEDVW,(SEQ ID NO: 265) RMKFDVWDLYFEEVW, (SEQ ID NO: 266) RMKFDVWDLYFEFVW,(SEQ ID NO: 267) RMKFDVWDLYFEGVW, (SEQ ID NO: 268) RMKFDVWDLYFEHVW,(SEQ ID NO: 269) RMKFDVWDLYFEKVW, (SEQ ID NO: 270) RMKFDVWDLYFELVW,(SEQ ID NO: 271) RMKFDVWDLYFEMVW, (SEQ ID NO: 272) RMKFDVWDLYFENVW,(SEQ ID NO: 273) RMKFDVWDLYFEPVW, (SEQ ID NO: 274) RMKFDVWDLYFEQVW,(SEQ ID NO: 275) RMKFDVWDLYFERVW, (SEQ ID NO: 276) RMKFDVWDLYFESVW,(SEQ ID NO: 277) RMKFDVWDLYFETVW, (SEQ ID NO: 278) RMKFDVWDLYFEVVW,(SEQ ID NO: 279) RMKFDVWDLYFEWVW, (SEQ ID NO: 280) RMKFDVWDLYFEYVW,(SEQ ID NO: 281) RMKFDVWDLYFEIAW, (SEQ ID NO: 282) RMKFDVWDLYFEICW,(SEQ ID NO: 283) RMKFDVWDLYFEIDW, (SEQ ID NO: 284) RMKFDVWDLYFEIEW,(SEQ ID NO: 285) RMKFDVWDLYFEIFW, (SEQ ID NO: 286) RMKFDVWDLYFEIGW,(SEQ ID NO: 287) RMKFDVWDLYFEIHW, (SEQ ID NO: 288) RMKFDVWDLYFEIIW,(SEQ ID NO: 289) RMKFDVWDLYFEIKW, (SEQ ID NO: 290) RMKFDVWDLYFEILW,(SEQ ID NO: 291) RMKFDVWDLYFEIMW, (SEQ ID NO: 292) RMKFDVWDLYFEINW,(SEQ ID NO: 293) RMKFDVWDLYFEIPW, (SEQ ID NO: 294) RMKFDVWDLYFEIQW,(SEQ ID NO: 295) RMKFDVWDLYFEIRW, (SEQ ID NO: 296) RMKFDVWDLYFEISW,(SEQ ID NO: 297) RMKFDVWDLYFEITW, (SEQ ID NO: 298) RMKFDVWDLYFEIWW,(SEQ ID NO: 299) RMKFDVWDLYFEIYW, (SEQ ID NO: 300) RMKFDVWDLYFEIVA,(SEQ ID NO: 301) RMKFDVWDLYFEIVC, (SEQ ID NO: 302) RMKFDVWDLYFEIVD,(SEQ ID NO: 303) RMKFDVWDLYFEIVE, (SEQ ID NO: 304) RMKFDVWDLYFEIVF,(SEQ ID NO: 305) RMKFDVWDLYFEIVG, (SEQ ID NO: 306) RMKFDVWDLYFEIVH,(SEQ ID NO: 307) RMKFDVWDLYFEIVI, (SEQ ID NO: 308) RMKFDVWDLYFEIVK,(SEQ ID NO: 309) RMKFDVWDLYFEIVL, (SEQ ID NO: 310) RMKFDVWDLYFEIVM,(SEQ ID NO: 311) RMKFDVWDLYFEIVN, (SEQ ID NO: 312) RMKFDVWDLYFEIVP,(SEQ ID NO: 313) RMKFDVWDLYFEIVQ, (SEQ ID NO: 314) RMKFDVWDLYFEIVR,(SEQ ID NO: 315) RMKFDVWDLYFEIVS, (SEQ ID NO: 316) RMKFDVWDLYFEIVT,(SEQ ID NO: 317) RMKFDVWDLYFEIVV, (SEQ ID NO: 318) RMKFDVWDLYFEIVY,(SEQ ID NO: 319) MKFDVWDLYFEIVW, (SEQ ID NO: 320) KFDVWDLYFEIVW.

Preferably, the peptide or peptide derivative of the second aspect ofthe invention comprises:

-   -   (i) an amino acid sequence comprising cimfwydcye; or    -   (ii) a variant amino acid sequence comprising one, two, three,        four, five, six or seven amino acid substitutions in cimfwydcye.

Preferably, at least one, two, three, four, five, six or seven of saidsubstitutions in cimfwydcye are D-amino acids.

Preferably, the peptide or peptide derivative of the second aspect ofthe invention comprises: an amino acid sequence comprisingX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀, wherein X₁, where present, is c, s, y, i, D-Pen,C, t, D-Nva, D-Nle or k, X₂ is i, y, w or d, X₃ is c or m, X₄ is f, t, vor c, X₅ is w or c, X₆ is y or c, X₇ is d, e or f, X₈ is c, e, f, y ord, X₉ is y or w and X₁₀ is e or i, with no more than seven amino acidssubstitutions compared to cimfwydcye.

Preferably, the peptide or peptide derivative comprises an amino acidsequence comprising X₁X₂X₃X₄wydX₈ye, wherein X₁ is c, C, D-Pen or s, X₂is l, y or w, X₃ is c or m, X₄ is f, t, or v and X₈ is c or e.

Preferably, the peptide or peptide derivative comprises an amino acidsequence comprising X₁X₂mX₄wydX₈ye, wherein X₁ is c, C or D-Pen, X₂ is ior y, X₄ is f, t, or v and X₈ is c or e.

Suitably, the peptide or peptide derivative of the second aspect of theinvention is a peptide or peptide derivative as represented in the tablebelow, or comprises or consists of the amino acid sequence of a peptideor peptide derivative as represented in tables 4 to 6 below:

TABLE 4 Most preferred peptides Peptide Sequence B03 Ac-cimfwydeye-NH₂B04 Disulphide-Dimer(Ac-cimfwydeye-NH₂)₂ B05 Ac-TTDS-(cymfwydc)-ye-NH₂B06 K-TTDS-(cymfwydc)-ye-NH₂ B14 Ac-(cimtwydc)-ye-NH₂ B15Ac-(cimvwydc)-ye-NH₂ B17 (cymfwydc)-ye B18 Ac-(cymfwydc)-yeG-NH₂ B19Ac-(D-Pen)imfwydeye-NH₂ B23 O(CH₂—CH₂—O—CH₂—CO-imfwydeye-NH₂)₂ B24Pyridine-3 (SEQ ID NO: 1), 5-(CO-imfwydeye-NH₂)₂ B34H₂N-E-TTDS-(cymfwydc)-ye-NH₂ B35 Ac-(cymfwydc)-yeK B37Ac-(cymfwydc)-ye-TTDS-K

In a preferred embodiment, peptides B05, B06, B14, B15, B17, B18, B34,B35 and B37 are cyclic.

TABLE 5 Preferred peptides Peptide Sequence B07 Ac-simfwydeye-NH₂ B07Ac-simfwydeye-NH₂ B09 Ac-ydmcwcefyi-NH₂ B10 Ac-idmccyfywe-NH₂ B16Ac-cimfwyddye-NH₂ B26 Ac-(cymfwydc)-ye B27 Ac-(cymfwydc)-ye-TTDS-NH₂ B28Ac-TTDS-(cymfwydc)-ye-TTDS-NH₂ B30 K-(cymfwydc)-ye-NH₂ B31Ac—K-(cymfwydc)-ye-NH₂ B32 E-(cymfwydc)-ye-NH₂ B33Ac—K-TTDS-(cymfwydc)-ye-NH₂ B36 Ac-(cymfwydc)-yeK—NH₂ B38Ac-(cymfwydc)-ye-TTDS-K—NH₂ B39 Ac-(cymfwydc)-ye-TTDS-E-NH₂ B41Ac-timfwydeye-NH₂

In a preferred embodiment, peptides B26, B27, B28, B30, B31, B32, B33,B36, B38 and B39 are cyclic.

TABLE 6 Active peptides Peptide Sequence B01 Ac-(cimfwydc)-ye-NH₂ B02Ac-(cymfwydc)-ye-NH₂ B11 Ac-(cwmfwydc)-ye-NH₂ B13 Ac-cicfwydcye-NH₂ B20Ac-(D-Nva)imfwydeye-NH₂ B21 Ac-(D-Nle)imfwydeye-NH₂ B22Ac-(Cys)imfwydeye-NH₂ B25 (cymfwydc)-ye-NH₂ B29TTDS-(cymfwydc)-ye-TTDS-NH₂ B40 Ac-kimfwydeye-NH₂

In a preferred embodiment, peptides B01, B02, B11, B25 and B29 arecyclic.

In the above tables, -TTDS- is 4,7,10-trioxa-1,13-tridecanediamine.“NH₂” is a C-terminal amide group.

B08 is deleted in the above tables, as being identical to B01. B12 isdeleted in the above tables, as being identical to B02.

Preferably, the peptide or peptide derivative of the first aspect of theinvention does not comprise or consist of a peptide represented in thelist below:

feiycwdcym, ywcfiymced, dmwceyfcyi, ceicwyfdym,ccwfiemdyy, cemdwycyfi, aimfwydcye, dimfwydcye,eimfwydcye, fimfwydcye, himfwydcye, iimfwydcye,kimfwydcye, limfwydcye, mimfwydcye, nimfwydcye,pimfwydcye, qimfwydcye, rimfwydcye, simfwydcye,timfwydcye, vimfwydcye, wimfwydcye, yimfwydcye,camfwydcye, ccmfwydsye, cdmfwydcye, cemfwydcye,cfmfwydcye, chmfwydcye, ckmfwydcye, clmfwydcye,cmmfwydcye, cnmfwydcye, cpmfwydcye, cqmfwydcye,crmfwydcye, csmfwydcye, ctmfwydcye, cvmfwydcye,ciafwydcye, cidfwydcye, ciefwydcye, ciffwydcye,cihfwydcye, ciifwydcye, cikfwydcye, cilfwydcye,cinfwydcye, cipfwydcye, ciqfwydcye, cirfwydcye,cisfwydcye, ciffwydcye, civfwydcye, ciwfwydcye,ciyfwydcye, cimawydcye, cimcwydsye, cimdwydcye,cimewydcye, cimhwydcye, cimiwydcye, cimkwydcye,cimlwydcye, cimmwydcye, cimnwydcye, cimpwydcye,cimqwydcye, cimrwydcye, cimswydcye, cimwwydcye,cimywydcye, cimfaydcye, cimfcydsye, cimfdydcye,cimfeydcye, cimffydcye, cimfhydcye, cimfiydcye,cimfkydcye, cimflydcye, cimfmydcye, cimfnydcye,cimfpydcye, cimfqydcye, cimfrydcye, cimfsydcye,cimftydcye, cimfvydcye, cimfyydcye, cimfwadcye,cimfwcdsye, cimfwddcye, cimfwedcye, cimfwfdcye,cimfwhdcye, cimfwidcye, cimfwkdcye, cimfwldcye,cimfwmdcye, cimfwndcye, cimfwpdcye, cimfwqdcye,cimfwrdcye, cimfwsdcye, cimfwtdcye, cimfwvdcye,cimfwwdcye, cimfwyacye, cimfwycsye, cimfwyecye,cimfwyfcye, cimfwyhcye, cimfwyicye, cimfwykcye,cimfwylcye, cimfwymcye, cimfwyncye, cimfwypcye,cimfwyqcye, cimfwyrcye, cimfwyscye, cimfwytcye,cimfwyvcye, cimfwywcye, cimfwyycye, cimfwydaye,cimfwydfye, cimfwydhye, cimfwydiye, cimfwydkye,cimfwydlye, cimfwydmye, cimfwydnye, cimfwydpye,cimfwydqye, cimfwydrye, cimfwydsye, cimfwydtye,cimfwydvye, cimfwydwye, cimfwydyye, cimfwydcae,cimfwydsce, cimfwydcde, cimfwydcee, cimfwydcfe,cimfwydche, cimfwydcie, cimfwydcke, cimfwydcle,cimfwydcme, cimfwydcne, cimfwydcpe, cimfwydcqe,cimfwydcre, cimfwydcse, cimfwydcte, cimfwydcve,cimfwydcwe, cimfwydcya, cimfwydsyc, cimfwydcyd,cimfwydcyf, cimfwydcyh, cimfwydcyi, cimfwydcyk,cimfwydcyl, cimfwydcym, cimfwydcyn, cimfwydcyp,cimfwydcyq, cimfwydcyr, cimfwydcys, cimfwydcyt,cimfwydcyv, cimfwydcyw, cimfwydcyy.

Preferably, the peptide or peptide derivative of the second aspect ofthe invention is a cyclic peptide. The peptide or peptide derivatives ofthe first aspect may also be cyclic.

The term “cyclic peptide” as used herein refers to a cyclic derivativeof a peptide to which, for example, two or more additional groupssuitable for cyclization have been added, often at the carboxyl terminusand at the amino terminus. Suitable groups include amino acid residues.A cyclic peptide may contain either an intramolecular disulfide bond,i.e. —S—S—, an intramolecular amide bond between the two added residues,i.e. —CONH— or —NHCO—, or intramolecular S-alkyl bonds, i.e.—S—(CH₂)n-CONH— or —NH—CO(CH₂)n-S—, wherein n is 1, 2 or more andpreferably no more than 6. Cyclization may be also carried out bytriazine chemistry as exemplified in Scham, D. et al. (2001) J. Org.Chem 66; 507. Cyclic peptide sequences are denoted with the prefix“cyclo” in front of the peptide sequence and the cyclic part of thesequence is incorporated in parenthesis and additionally separated fromthe rest of the sequence by hyphens.

A peptide or peptide derivative of the first or second aspect of theinvention may be modified by conjugation to polyethylene glycol (PEG).Suitable methods of PEGylation are disclosed in U.S. Pat. No. 5,122,614(Zalipsky; Enzon, Inc.) and U.S. Pat. No. 5,539,063 (Hakimi et al;Hoffmann-La Roche Inc.), all of which PEGylation methods areincorporated herein by reference. Various molecular weights of PEG maybe used, suitably from 5000 to 40000 kD. A preferred molecular weight is5000 kD. Preferably, the PEG is monodisperse, meaning that there islittle variation in molecular weight between PEG molecules. PEGylationmay improve the solubility and plasma half-life of a peptide.

A third aspect of the invention provides a dual peptide comprising apeptide or peptide derivative of the first or second aspects of theinvention conjugated to a further peptide or peptide derivative of thefirst or second aspects of the invention, wherein the peptide or peptidederivative may be the same as or different from the further peptide orpeptide derivative, and wherein the dual peptide has procoagulantactivity.

The dual peptide may comprise two of the same, or two different,peptides or peptide derivatives of the first or second aspects of theinvention covalently linked to one another, either by a flexible linkerwhich can be peptidic, peptidomimetic or non-peptidic, or by aconformationally constrained linker that can comprise conformationallyconstrained peptidic, peptidomimetic or non-peptidic building blockse.g. triazine moieties, or by any other possible method known in theart.

Preferably, the peptide or peptide derivative of the first and secondaspects of the invention and the dual peptide of the third aspect of theinvention has a molecular weight of between 0.5 and 3.5 kD. By“molecular weight” we mean the theoretical mass of a monomer of thepeptide or peptide derivative exclusive of any counter ions or adducts.For PEGylated peptides the molecular weight is defined as the mass ofthe monomeric molecule exclusive of any counter ions or adducts andexclusive of the PEG moiety or moieties. Peptides, peptide derivativesand dual peptides of between 0.5 kD and 3.5 kD are more readilysynthesised than larger peptides, have a reduced risk being immunogenic,and are generally easily administered to a patient. Peptides of lessthan 0.5 kD may be readily synthesised and administered and are lesslikely to be immunogenic, but may not possess the required procoagulantactivity. Nevertheless, peptides, peptide derivatives and dual peptidesof less than 0.5 kD and greater than 3.5 kD are encompassed by theinvention if they possess the appropriate activity.

The peptides and peptide derivatives of the first and second aspects ofthe invention and the dual peptide of the third aspect of the inventionpossess procoagulant activity.

By “procoagulant activity” we mean the ability to promote thrombingeneration and/or fibrin deposition in a suitable test system.

It will be appreciated that different assays are available to determineprocoagulant activity. Indeed, there are different types of procoagulantactivity. Peptides and peptide derivatives may promote coagulation inplasma depleted of FV, FVII, FVIII, FX or FXI. In a preferredembodiment, a peptide or peptide derivative of the invention promotesthrombin generation and/or fibrin deposition in plasma in which FVIII isdepleted or absent. This type of activity is referred to as coagulationFVIII activity. Where the plasma is from an individual lacking FVIII,the activity is typically referred to as FVIII equivalent activity.Where the plasma contains inhibitors against FVIII, the activity istypically referred to as FVIII inhibitor bypassing equivalent activity.Other procoagulant activities include FV activity, FVII activity, FXactivity and FXI activity.

Individual peptides and peptide derivatives may vary in their relativeefficacy between different types of assay. Therefore, even if a peptideor peptide derivative appears to have a low efficacy in a particularassay, it may nevertheless possess a suitably high level of procoagulantactivity in another assay.

A suitable assay to determine procoagulant activity is the DefinedIntrinsic Thrombin Generation Assay described below. In this assay, acompound is considered to have procoagulant activity if, at aconcentration of 25, 50 or 100 μM it can stimulate the generation of 5nM thrombin in 60 minutes, and preferably in 50, 40, 30, 20 or 10minutes. Preferably, it can stimulate generation of 10 nM thrombin in 60minutes, and more preferably in 50, 40, 30, 20 or 10 minutes. Analternative assay is the Defined Dual-Pathway Thrombin Generation Assaydescribed below. In this assay, a compound is considered to haveprocoagulant activity if, at a concentration of 25, 50 or 100 μM it canstimulate the generation of 5 nM thrombin in 70 minutes, and preferably60, 50, 40, 30 or 20 minutes. Preferably, it can stimulate generation of10 nM thrombin in 70 minutes, and more preferably 60, 50, 40, 30 or 20minutes. The above assays are particularly useful for determiningcoagulation FVIII activity because they are conducted in the presence ofFVIII-depleted or inhibited plasma. However, they can be readily adaptedto test for other types of procoagulant activity by substituting asuitable depleted or inhibited plasma for FVIII-depleted or inhibitedplasma.

Suitably, the procoagulant activity is a thrombin generation time of 25,50 or 100 μM of compound in a Defined Intrinsic Thrombin GenerationAssay equivalent to that of at least 100 mU/mL Factor Eight InhibitorBypassing Activity (FEIBA), preferably at least 300 mU/mL FEIBA, morepreferably at least 600 mU/mL FEIBA and most preferably at least 1200mU/mL FEIBA. Thrombin generation time or peak time is the time intervalfrom the addition of the pre-warmed plasma to the other components inthe assay described below, to the time of the thrombin peak maximum.

Alternatively, the procoagulant activity is a thrombin peak maximum of25, 50 or 100 μM of compound in a Defined Dual-Pathway ThrombinGeneration Assay (DDPTGA) equivalent to at least 1 mU/mL Factor EightInhibitor Bypassing Activity (FEIBA), preferably at least 5 mU/mL FEIBA,most preferably at least 10 mU/mL FEIBA. Thrombin peak maximum, alsoreferred to as Peak IIa is the maximal thrombin concentration generatedduring the assay. The Defined Dual-Pathway Thrombin Generation Assay canbe used to determine coagulation activities other than FVIII activity ifsuitable factor depleted plasma is substituted for FVIII deficient orinhibited plasma. A peptide, peptide derivative or dual peptide of theinvention is considered to have FV, FVII, FX or FXI activity if, at aconcentration of 25, 50 or 100 μM, it can stimulate the generation ofmore thrombin in a DDPTGA using FV, FVII, FX or FXI deficient plasmarespectively over 120 minutes than is stimulated in the absence ofpeptide.

Suitably, the procoagulant activity is a thrombin generation time of 25,50 or 100 μM of compound in a Defined Intrinsic Thrombin GenerationAssay peaking within 30 minutes, preferably within 15 minutes and mostpreferably within 10 minutes. [0]Alternatively, the procoagulantactivity is a thrombin generation time of 25, 50 or 100 μM of compoundin a Defined Dual-Pathway Thrombin Generation Assay peaking within 50minutes, preferably within 45 minutes and most preferably within 30minutes.

The effect of a peptide or peptide derivative or dual peptide onthrombin generation may be determined in FVIII immuno inhibited, FVIIIimmuno depleted, FVIII inhibitor patient or hemophilia A patient plasmaor other types of coagulation factor deficient plasmas, for example bycontinuously monitoring the slow cleavage of the thrombin-specificfluorogenic substrate I-1140 (Bachem) in a black 96-well micro plate(Cliniplate, Thermo Labsystems) as described below. Parameters that canusefully be measured in thrombin generation assays to determine theeffect of the peptide or peptide derivative are thrombin concentrationat peak time; thrombin generation time at peak thrombin; slope ofpropagation phase of thrombin generation curve and lag time of thrombingeneration (initiation phase).

The intrinsic pathway of thrombin generation may be assayed in athrombin generation assay by including FXIa and phospholipids. In suchan assay, which is similar to an activated partial thromboplastin time(aPTT) test, thrombin generation is solely directed through theintrinsic pathway, and is FVIII dependent. A suitable assay is theDefined Intrinsic Thrombin Generation Assay described below.Alternatively, by employing low concentrations of TF and phospholipidsinstead of FXIa and phospholipids, thrombin is generated by both theextrinsic (tissue factor) and the intrinsic pathways. This form of thethrombin generation assay is the more physiologic one, as both thrombingeneration pathways are involved; it is partially FVIII dependent. Asuitable assay is the Defined Dual-Pathway Thrombin Generation Assay.

The Defined Intrinsic Thrombin Generation Assay is performed as follows.FVIII activity of human plasma is inhibited by incubating (2 hours, 37°C.) 40 μl of human normal plasma with 10 μl heat inactivated anti-humanFVIII plasma raised in goat (600 BU/ml, 6 hours incubated at 56° C.). A15 μl mix of FXIa (16.67 nM) (Enzyme Research Laboratories) andphospholipids (Phosphatidylcholine/Phosphatidylserine 60%/40%, 120 μM)(Avanti Polar Lipids), 15 μl mix of 3.33 mM I-1140 and 50 mM CaCl₂ and10 μl peptide solution (different concentrations) are added to 10 μl 2×HNa/HSA5 (50 mM Hepes, 350 mM NaCl, pH7.35, 10 mg/ml HSA). After sixminutes incubation at 37° C., thrombin generation is started by theaddition of 50 μl pre-warmed (37° C.) FVIII-inhibited plasma. Instead ofFVIII-inhibited plasma, FVIII inhibitor patient plasma or severaldepleted plasmas can be used. The micro-plate is immediately put into aGENios Plus (Tecan) or Safire 2 (Tecan) fluorescence reader and thefluorescence signal (ex 340 nm/em 440 nM) is followed kinetically byreading the plate every 21 seconds. By deviating the originalfluorescence data the amount of generated thrombin is calculated from astandard curve constructed using a concentration range of thrombin.

For calculation of activity equivalent units experiments are performedwith dilutions of Factor Eight Inhibitor Bypassing Agent (FEIBA, BaxterAG), Immunate (human FVIII, purified plasma derived) reference standard(Baxter AG) or Recombinate standard (human FVIII, purified recombinant,Baxter AG). A linear fit of the logarithm of FEIBA (FVIII) concentrationplotted against thrombin generation time at peak thrombin results in astandard curve. With this curve FEIBA (FVIII) equivalent activity iscalculated for a defined peptide concentration.

Where a peptide concentration is given herein, it is to be understoodthat it is not the concentration of peptide in the final assay volume,but a concentration as corrected for plasma volume. The concentration inthe final assay volume is the corrected concentration divided by 2.5.Thus, where a concentration of 100 μM is given, the actual concentrationin the final assay volume in 40 μM. Similarly, the FEIBA equivalentactivity is also corrected for plasma volume. Thus, if it is stated thatat 100 μM a peptide has an activity equivalent to 100 mU/ml FEIBA in theDITGA, the concentration of peptide in the final assay volume is 40 μMand the equivalent concentration of FEIBA in the control assay is 40mU/ml FEIBA.

The Defined Dual-Pathway Thrombin Generation Assay is performed asdescribed below, using a commercial test kit (Technothrombin TGA,Technoclone GmbH, Vienna, Austria). Briefly, a mix of 40 μl 1.25 mMfluorogenic substrate (Z-GGR-AMC) 18.75 mM CaCl₂, 10 μl TGA reagent B(phospholipid vesicles Phosphatidylcholine/Phosphatidylserine 80%/20%(3.2 μM) containing 17.9 pM recombinant human tissue factor; TechnocloneGmbH) or 10 μl TGA reagent C high (phospholipid vesiclesPhosphatidylcholine/Phosphatidylserine 80%/20% (32 μM) containing 71.6pM recombinant human tissue factor; Technoclone GmbH) and 10 μl peptidedilution, FEIBA reference standard or FVIIa standard dilutions (EnzymeResearch Laboratories, South Bend, Ind. USA) are incubated four minutesat 37° C. Preferably, Reagent C high is used. Thrombin generation isstarted by the addition of 40 μl of one of several types of human plasma(37° C.). Conversion of the fluorogenic substrate by thrombin isfollowed by immediately putting the plate into a preheated (37° C.)microplate fluorescence reader (Tecan Safire 2, ex 360 nm/em 460 nm) andkinetically reading the plate every 30 seconds. By deviating theoriginal fluorescence data the amount of generated thrombin iscalculated from a standard curve constructed using a concentration rangeof thrombin. Non linear regression analysis of factor VIIa or FEIBAconcentrations plotted against the thrombin at peak of the thrombingeneration curve or time to peak thrombin results in standard curves.With these curves, factor VIIa or FEIBA equivalent activity can becalculated for a defined peptide concentration. As described in relationto DITGA, where a peptide concentration is given herein in relation tothe DDPTGA, it is to be understood that it is not the concentration ofpeptide in the final assay volume, but a concentration as corrected forplasma volume. The concentration in the final assay volume is thecorrected concentration divided by 2.5. FEIBA equivalent activity isalso corrected for plasma volume by applying the same correction factor.

Another suitable assay to determine procoagulant activity, andparticularly FVIII equivalent activity or FVIII inhibitor bypassingactivity, is the Defined Fibrin Deposition Assay as described below.Suitably, the procoagulant activity of a sample of 25 μM test compoundin the Defined Fibrin Deposition Assay is equivalent to at least 30mU/mL Factor Eight Inhibitor Bypassing Activity (FEIBA), preferably atleast 80 mU/mL FEIBA, most preferably at least 200 mU/mL FEIBA. Thisassay is particularly useful for determining coagulation FVIII activitybecause it is conducted in the presence of FVIII-depleted or inhibitedplasma.

The Defined Fibrin Deposition Assay is performed as follows. FVIIIactivity of human citrated plasma (Baxter AG) is first inhibited byincubating (2 hours, 37° C.) 100 μl of human normal plasma with 25 μlheat inactivated anti-human FVIII plasma (300 BU/ml, 6 hours incubatedat 56° C.) raised in goat. For each sample to be tested, 125 μl of thisFVIII-inhibited human normal plasma is transferred to a pre-warmedcuvette and a 75 μl dilution of a test compound or FEIBA referencestandard (Baxter AG) is added. The dilutions of test compound or FEIBAreference standard contain 50 mM imidazole, 100 mM NaCl and 10 mg/mlhuman serum albumin (Sigma) pH 7.4. As a trigger and for providingprocoagulant surfaces 100 μl of a mix of human factor XIa (3.13 nM,Enzyme Research Laboratories) and phospholipid (PL) vesicles(Phosphatidylcholine/Phosphatidylserine 60%/40%, 30 μM; Avanti PolarLipids) in 50 mM Imidazole, 100 mM NaCl, 10 mg/ml human serum albumin(Sigma) pH 7.4 is included. After incubating for three minutes at 37° C.the coagulation reaction is started by adding 100 μl of 25 mM CaCl₂.Clot formation is monitored by a coagulometer (KC10A, Amelung, Germany).In brief, each cuvette rotates slowly above the magnetic detectiondevice and contains a small magnetic metallic ball. Whilst the plasmacomponents remain in solution, the ball sits at the bottom of thecuvette. Over time, a clot begins to form, such that the ball starts torotate with the developing clot in the rotating cuvette. The “clottingtime” is recorded and is defined as the time from addition of the CaCl₂to the time that the developing clot begins to rotate the metallic ball.A standard curve for FEIBA reference standard dilutions is calculated bylinear regression of logarithmic FEIBA concentrations (x-axis) againstthe clotting time (y-axis). Based on the clotting time of each compoundconcentration FEIBA equivalent activities are calculated according tothis standard curve.

Where a peptide concentration is given herein in relation to the DefinedFibrin Deposition Assay, it is to be understood that it is not theconcentration of peptide in the final assay volume, but a concentrationas corrected for plasma volume. The concentration in the final assayvolume is the corrected concentration divided by 4. Thus, where aconcentration of 100 μM is given, the actual concentration in the finalassay volume in 25 μM. Similarly, the FEIBA equivalent activity is alsocorrected for plasma volume. Thus, if it is stated that at 100 μM apeptide has an activity equivalent to 100 mU/ml FEIBA in the DefinedFibrin Deposition Assay, the concentration of peptide in the final assayvolume is 25 μM and the equivalent concentration of FEIBA in the controlassay is 25 mU/ml FEIBA.

Preferably, the peptides and peptide derivatives of the first and secondaspects of the invention and the dual peptide of the third aspect of theinvention can at least partially compensate for the absence ofbiologically active FVIII when administered in an animal model of severehuman hemophilia A. For example, they may be active in controllingbleeding in FVIII deficient mice, such as the strains described indetail by Bi et al (Nat Genet. 1995; 10:119-21), in which exon 17 orexon 16 of FVIII is disrupted. The exon 16 FVIII−/− mice are availablefrom Jackson Laboratory, 600 Main Street, Bar Harbor, Me. 04609 USA(strain name: B6; 129S4-F8^(tm1Kaz)/J).

A suitable assay to test the ability of a compound to control bleedingis the tail clip assay. Peptides, peptide derivatives or dual peptidesare administered to mice in a suitable vehicle, typically i.v., i.p. ors.c. Different doses of each peptide or peptide derivative may beadministered to different groups of mice to determine dose-dependency.Groups of mice, typically 8-16 male and female exon 17 FVIII knockoutmice with severe hemorrhagic diathesis, receive a single i.v. (tailvein), i.p. or s.c. bolus injection (10 ml/kg body weight). Two minutesbefore tail clip, animals are anesthetized by an i.p. application of 100mg/kg ketamine and 5 mg/kg xylazine. Five minutes after i.v. and 60minutes after i.p. or s.c. peptide or peptide derivative administration0.5 cm of the tail tip is ablated. Blood dropping from the wound iscollected in tubes containing 5.0 ml 0.04% NH₃ for defined time periods,such as 0-2 minutes, 2-4 minutes, 4-6 minutes, 6-8, 8-10, 10-12, 12-14,14-16, 16-20, 20-24, 24-28, 28-32, 32-42, 42-52 and 52-62 minutes. Bloodcells in each tube are disrupted and hemoglobin is extracted by a threehour incubation period at room temperature followed by ultrasoundtreatment. The absorbance at 414 nm and 620 nm of the extracts isdetermined in micro titre plates. 620 nm is a reference wavelength andthe A₆₂₀ reading is subtracted from the A₄₁₄ reading. The amount ofblood in the extract corresponding to the subtracted reading iscalculated from a standard curve created by known amounts of blood fromwild type control mice, such as C57/Bl6 mice. Parameters of the bleedingcharacteristics of the mice to be recorded are total blood loss,bleeding rate, bleeding time, 1 h, 2 h, 3 h, 4 h, 24 h and 48 hsurvival. Cumulative blood loss is calculated by summing up the amountsof blood for each time period. Data for the animals of a group areaveraged and plotted against bleeding time. At each time point data setsfor treatment and vehicle control groups are analysed by Student'st-test for statistical significance.

Preferably, mice administered the peptide, peptide derivative or dualpeptide have a blood loss in the tail clip assay at 62 minutes from tailclip of no more than 70% of the blood loss of mice administered thevehicle alone, more preferably no more than 60% and most preferably nomore than 50% of the blood loss of mice administered the vehicle alone.

Preferably, survival of mice administered the peptide, peptidederivative or dual peptide in the above assay is at least 40%, morepreferably at least 60% and most preferably at least 80% at 2 hoursafter tail clip. Preferably, survival of mice administered the peptideor peptide derivative in the tail clip assay is at least 20%, morepreferably at least 30% and most preferably at least 40% at 24 hoursafter tail clip.

Preferably, the peptide or peptide derivative of the first and secondaspects of the invention or the dual peptide of the third aspect of theinvention has a stability in human plasma at 30 minutes of at least 50%,preferably at least 70%, more preferably at least 80% and mostpreferably at least 90%. A suitable assay to determine stability inhuman plasma is described in the Examples.

Preferably, the peptide or peptide derivative of the first and secondaspects of the invention or the dual peptide of the third aspect of theinvention has an aqueous solubility in phosphate buffered saline pH 7.4at 25° C. of at least 25 μM, preferably at least 60 μM and mostpreferably at least 100 μM. A suitable assay to determine aqueoussolubility in phosphate buffered saline pH 7.4 at 25° C. is described inthe Examples.

Herein, the term “Factor VIII” or “FVIII” refers to any FVIII moietywhich exhibits biological activity that is associated with native FVIII.The sequence of FVIII can be found as NCBI Accession Number NP_000123 orUniProtKB/Swiss-Prot entry P00451.

As used herein, “plasma-derived FVIII” includes all forms of the proteinfound in blood obtained from a mammal having the property of activatingthe coagulation pathway.

As used herein, “rFVIII” denotes FVIII obtained via recombinant DNAtechnology.

A fourth aspect of the invention provides a pharmaceutical compositioncomprising the peptide or peptide derivative of the first or secondaspects of the invention or the dual peptide of the third aspect of theinvention. Peptides, peptide derivatives and dual peptides may be in theform of pharmaceutically acceptable salts, solvates or hydrates.Suitably, the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier. The carrier may be preferably a liquid formulation,and is preferably a buffered, isotonic, aqueous solution. Suitably, thepharmaceutical composition has a pH that is physiologic, or close tophysiologic. Suitably it is of physiologic or close to physiologicosmolarity and salinity. It may contain sodium chloride and/or sodiumacetate. The peptides, peptide derivatives and dual peptides of theinvention can be made without significant pyrogenicity that might occurin production of biological treatments. This can be important,especially for intravenous formulations where only low levels ofendotoxin can be tolerated. It is preferred that subcutaneous,intraperitoneal, buccal, intravenous and other parenteral formulationsare sterile and endotoxin free.

Pharmaceutically acceptable carriers may also include excipients, suchas diluents, and the like, and additives, such as stabilizing agents,preservatives, solubilizing agents, and the like. The peptides of thisinvention may be also in the form of any pharmaceutically acceptablesalt.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of US or EU or other government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inhumans.

The composition can be also for example a suspension, emulsion,sustained release formulation, cream, gel or powder. The composition canbe formulated as a suppository, with traditional binders and carrierssuch as triglycerides.

Though intravenous delivery of the peptides, peptide derivatives anddual peptides of the present invention may be possible a non-intravenousroute is preferred, particularly subcutaneous, nasal, buccal, oral orpulmonary delivery. Intraperitoneal (i.p.) delivery may also be used.

Pharmaceutical compositions may additionally comprise, for example, oneor more of water, buffers (e.g., neutral buffered saline or phosphatebuffered saline), ethanol, mineral oil, vegetable oil,dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, adjuvants, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione and/or preservatives. Furthermore, one or more other activeingredients may (but need not) be included in the pharmaceuticalcompositions provided herein.

Pharmaceutical compositions may be formulated for any appropriate mannerof administration, including, for example, topical (e.g., transdermal orocular), oral, buccal, nasal, vaginal, rectal or parenteraladministration. The term parenteral as used herein includessubcutaneous, intradermal, intravascular (e.g., intravenous),intramuscular, spinal, intracranial, intrathecal, intraocular,periocular, intraorbital, intrasynovial and intraperitoneal injection,as well as any similar injection or infusion technique. Forms suitablefor oral use include, for example, tablets, troches, lozenges, aqueousor oily suspensions, dispersible powders or granules, emulsion, hard orsoft capsules, or syrups or elixirs. Compositions provided herein may beformulated as a lyophilizate.

Aqueous suspensions contain the active ingredient(s) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions may also comprise one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Peptides or peptide derivatives may be formulated for local or topicaladministration, such as for topical application to the skin, wounds ormucous membranes, such as in the eye. Formulations for topicaladministration typically comprise a topical vehicle combined with activeagent(s), with or without additional optional components. Suitabletopical vehicles and additional components are well known in the art,and it will be apparent that the choice of a vehicle will depend on theparticular physical form and mode of delivery. Topical vehicles includewater; organic solvents such as alcohols (e.g., ethanol or isopropylalcohol) or glycerin; glycols (e.g., butylene, isoprene or propyleneglycol); aliphatic alcohols (e.g., lanolin); mixtures of water andorganic solvents and mixtures of organic solvents such as alcohol andglycerin; lipid-based materials such as fatty acids, acylglycerols(including oils, such as mineral oil, and fats of natural or syntheticorigin), phosphoglycerides, sphingolipids and waxes; protein-basedmaterials such as collagen and gelatin; silicone-based materials (bothnon-volatile and volatile); and hydrocarbon-based materials such asmicrosponges and polymer matrices. A composition may further include oneor more components adapted to improve the stability or effectiveness ofthe applied formulation, such as stabilizing agents, suspending agents,emulsifying agents, viscosity adjusters, gelling agents, preservatives,antioxidants, skin penetration enhancers, moisturizers and sustainedrelease materials. Examples of such components are described inMartindale—The Extra Pharmacopoeia (Pharmaceutical Press, London 1993)and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations maycomprise microcapsules, such as hydroxymethylcellulose orgelatin-microcapsules, liposomes, albumin microspheres, microemulsions,nanoparticles or nanocapsules.

A pharmaceutical composition may be formulated as inhaled formulations,including sprays, mists, or aerosols. For inhalation formulations, thecompounds provided herein may be delivered via any inhalation methodsknown to those skilled in the art. Such inhalation methods and devicesinclude, but are not limited to, metered dose inhalers with propellantssuch as CFC or HFA or propellants that are physiologically andenvironmentally acceptable. Other suitable devices are breath operatedinhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosolformulations for use in the subject method typically includepropellants, surfactants and co-solvents and may be filled intoconventional aerosol containers that are closed by a suitable meteringvalve.

Inhalant compositions may comprise liquid or powdered compositionscontaining the active ingredient that are suitable for nebulization andintrabronchial use, or aerosol compositions administered via an aerosolunit dispensing metered doses. Suitable liquid compositions comprise theactive ingredient in an aqueous, pharmaceutically acceptable inhalantsolvent, e.g., isotonic saline or bacteriostatic water. The solutionsare administered by means of a pump or squeeze-actuated nebulized spraydispenser, or by any other conventional means for causing or enablingthe requisite dosage amount of the liquid composition to be inhaled intothe patient's lungs. Suitable formulations, wherein the carrier is aliquid, for administration, as for example, a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.

Formulations or compositions suitable for nasal administration, whereinthe carrier is a solid, include a coarse powder having a particle size,for example, in the range of 20 to 500 microns which is administered inthe manner in which snuff is administered (i.e., by rapid inhalationthrough the nasal passage from a container of the powder held close upto the nose). Suitable powder compositions include, by way ofillustration, powdered preparations of the active ingredient thoroughlyintermixed with lactose or other inert powders acceptable forintrabronchial administration. The powder compositions can beadministered via an aerosol dispenser or encased in a breakable capsulewhich may be inserted by the patient into a device that punctures thecapsule and blows the powder out in a steady stream suitable forinhalation.

Pharmaceutical compositions may be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of modulator following administration). Such formulations maygenerally be prepared using well known technology and administered by,for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofmodulator release. The amount of modulator contained within a sustainedrelease formulation depends upon, for example, the site of implantation,the rate and expected duration of release and the nature of thecondition to be treated or prevented.

Pharmaceutical compositions may be formulated with an agent to improvebioavailability, such an as organic solvent. For example, Cremophor EL®(Product No. 00647/1/63; BASF Aktiengesellschaft, Germany) is apolyethoxylated castor oil which is prepared by reacting 35 moles ofethylene oxide with each mole of castor oil. It may be used to stabiliseemulsions of non-polar materials in aqueous systems. Alternatively,peptide, peptide derivative or dual peptide may be incorporated withinor bound to a proteinaceous micro or nano-particle for improvedbioavailability. Suitable micro- and nano-particles are described inU.S. Pat. No. 5,439,686 (Desai et al; Vivorx Pharmaceuticals, Inc., CA)and U.S. Pat. No. 5,498,421 (Grinstaff et al; Vivorx Pharmaceuticals,Inc., CA). Suitably, the proteinaceous nano-particle comprises humanserum albumin, particularly human serum albumin or a recombinant formthereof. WO 2007/077561 (Gabbai; Do-Coop Technologies Ltd., Israel)describe another suitable carrier comprising nanostructures and aliquid, referred to therein as Neowater™.

For oral and parenteral administration to patients, including humanpatients, the daily dosage level of the peptide, peptide derivative ordual peptide of the invention will usually be from 2 to 2000 mg peradult (i.e. from about 0.03 to 30 mg/kg), administered in single ordivided doses.

Thus, for example, the tablets or capsules of the peptide, peptidederivative or dual peptide of the invention may contain from 2 mg to2000 mg of active compound for administration singly or two or more at atime, as appropriate. The physician in any event will determine theactual dosage which will be most suitable for any individual patient andit will vary with the age, weight and response of the particularpatient. The above dosages are exemplary of the average case. There can,of course, be individual instances where higher or lower dosage rangesare merited and such are within the scope of this invention.

For veterinary use, a peptide, peptide derivative or dual peptide of theinvention is administered as a suitably acceptable formulation inaccordance with normal veterinary practice and the veterinary surgeonwill determine the dosing regimen and route of administration which willbe most appropriate for a particular animal.

The peptides, peptide derivatives and dual peptides disclosed herein canbe used for medical applications and animal husbandry or veterinaryapplications. Typically, the product is used in humans. The term“patient” is intended to denote a mammalian individual, and is so usedthroughout the specification and in the claims.

A fifth aspect of the invention provides a peptide or peptide derivativeof the first or second aspects or a dual peptide of the third aspect ofthe invention for treating a patient having a deficiency in FV, FVII,FVIII, FX and/or FXI.

A sixth aspect of the invention provides a use of a peptide or peptidederivative of the first or second aspects or a dual peptide of the thirdaspect of the invention in the manufacture of a medicament for thetreatment of a deficiency in FV, FVII, FVIII, FX and/or FXI in apatient.

An seventh aspect of the invention provides a method of treating apatient having a deficiency in FV, FVII, FVIII, FX and/or FXI comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition of the fourth aspect.

The peptides, peptide derivatives and dual peptides of the presentinvention can be used for the treatment of a deficiency in FV, FVII,FVIII, FX and/or FXI for both the prophylaxis and for treatment of acutebleeds. Patients with FVIII deficiency (hemophilia A) often developinhibitor antibodies to FVIII. Inhibitor development (to FIX) is alsoknown in FIX deficiency (hemophilia B). Since FV, FVII, FXI and FXdeficiencies are very rare congenital disorders little is known aboutinhibitor development, although it is feasible that patients having suchdisorders might develop inhibitors. Treatment of inhibitor patients is apreferred embodiment of the fifth, sixth and seventh aspects. Suchinhibitor patients may have either a high titer response of greater than5 B U or a low titer response of between 0.5 and 5 BU. Typically, theinhibitors are directed against FVIII and the patients have hemophiliaA.

The magnitude of the antibody response to FVIII can be quantified usinga functional inhibitor assay, such as that described in [0]Kasper C K etal (1975) Proceedings: A more uniform measurement of factor VIIIinhibitors. Thromb Diath Haemorrh. 34(2):612. FXI inhibitors could bequantified by an aPTT assay as described by Kasper. Inhibitors of FV,FVII and FX could be quantified by a PT based assay following theprocedure of Kasper.

A peptide or peptide derivative according to the eighth, ninth or tenthaspects of the invention is not FVIII or a fragment thereof. Typically,it does not consist of or comprise the amino acid sequence of any FVIIIprotein, whether of human, mammalian or vertebrate origin. Neither doesit consist of a fragment of a FVIII protein. Typically, it comprisesfewer than 50, fewer than 20, fewer than 10, fewer than 5 contiguousamino acids of a FVIII protein, such as a human FVIII protein. Preferredpeptides and peptide derivatives are the peptides and peptidederivatives of the first and second aspects of the invention, or thedual peptides of the third aspect of the invention. Alternative peptidesand peptide derivative may be synthesised and tested for procoagulantactivity as described in relation to the exemplified peptides andpeptide derivatives.

Peptides and peptide derivatives of the eighth, ninth or tenth aspectsof the invention may be formulated as pharmaceutical compositions, asdescribed above, and may be used in medicine as described above.

The present invention will be further illustrated in the followingexamples, without any limitation thereto.

Example 1: Synthesis and Identification of Compounds with ThrombinGenerating Activity

Compounds were screened using the “Defined intrinsic thrombin generationassay” in which thrombin generation was quantified in vitro inFVIII-inhibited human plasma in the presence of Factor XIa andphospholipid vesicles. Further compounds were screened in the aboveassay, and in the “Defined dual-pathway thrombin generation assay” usingtissue factor and phospholipids instead of Factor XIa and phospholipids,as described in the specific description.

The compounds, which are peptides and peptide derivatives, weresynthesised by classical solid phase peptide synthesis or SPOT-Synthesisat 50-100 nmol peptide per spot, which allows positionally addressable,chemical synthesis of peptides on continuous cellulose membranes.Peptides were dissolved in either 10% or 50% DMSO in water.

PEGylation of peptides and peptide derivatives was carried out asfollows. PEG5000 NHS-ester was coupled to the N-terminus of theHPLC-purified peptides in solution. If lysine was present in the peptidesequence, this amino acid was protected with the ivDde protecting groupin order to avoid PEGylation at the ε-amino group. After coupling of thePEG5000 to the N-terminus, the ivDde protecting group was cleaved off by3% hydrazine hydrate in dimethyl formamide followed by repurification ofthe final product by HPLC.

Compounds deemed to promote thrombin generation were identified, asindicated in Tables 7 and 8 below.

TABLE 7 Compounds based on A01 Peptide Sequence SEQ ID NO: 2 A01Ac-RMKFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A02 Ac-PMKFDVWDLYFEIVW-NH₂SEQ ID NO: 2 A03 Ac-RMDFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A04Ac-RMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 1 A05 Ac-WDLYFEIVW-NH₂ SEQ ID NO: 3A06 Ac-WDLYFEIVWE SEQ ID NO: 1 A07 Ac-WDLYFEIVW-O-E SEQ ID NO: 2 A08O-RMEFDVWDLYFEIVW-O-NH₂ SEQ ID NO: 4 A09 ERMEFDVWDLYFEIVW-NH₂SEQ ID NO: 34 A10 EPMKFDVWDLYFEIVW-NH₂ SEQ ID NO: 2 A11O-RMDFDVWDLYFEIVW-O-NH₂ SEQ ID NO: 5 A12 ERXEFDVWDLYFEIVW-NH₂ X is NvaA13 O-RMEFDVWDLYXEIVW-O-NH₂ X is Phg SEQ ID NO: 6 A14 Ac-WSLYFEIVWESEQ ID NO: 1 A15 Ac-WDLYFEISW-O-E SEQ ID NO: 2 A16PEG5000-RMKFDVWDLYFEIVW-NH₂ SEQ ID NO: 6 A17 PEG5000-WSLYFEIVWESEQ ID NO: 4 A18 PEG5000-ERMEFDVWDLYFEIVW-NH₂ SEQ ID NO: 7 A19Ac-VWDLYFEIVW-NH₂ SEQ ID NO: 1 A20 Ac-WDLYFEIVW-O-K

In the above table, O— is 4,7,10-trioxa-1,13-tridecanediamine (ttds)

TABLE 8 Compounds based on B01 Peptide Sequence B01 Ac-(cimfwydc)-ye-NH₂B02 Ac-(cymfwydc)-ye-NH₂ B03 Ac-cimfwydeye-NH₂ B04Disulphide-Dimer(Ac-cimfwydeye-NH₂)₂ B05 Ac—O-(cymfwydc)-ye-NH₂ B06K—O-(cymfwydc)-ye-NH₂ B07 Ac-simfwydeye-NH₂

In the above table, —O— is 4,7,10-trioxa-1,13-tridecanediamine (ttds).The actual peptides used in this study, designated B01, B02, B05 andB06, were cyclic.

Example 2: Testing of Compounds in Intrinsic and Dual-Pathway ThrombinGeneration Assays

Various concentrations of each peptide were tested in the definedintrinsic thrombin generation assay using human FVIII inhibited plasma.Results are given in the table below.

TABLE 9 Activity of peptide compounds in the defined intrinsic thrombingeneration assay (triggered by FXIa). Thrombin peak time is given asFEIBA equivalent activity (mU/ml) calculated based on a FEIBA standardcalibration curve. Concentration (μM) Compound 200 100 50 25 12.5 6.25A01 338 227 160 138 97 A03 1396 1069 762 639 477 A05 816 631 562 487 390A19 826 663 525 495 383 B01 103 89 62 35 A02 1305 1018 780 674 577 B031394 1030 738 602 454 B05 1089 649 270 152 126 B06 902 378 172 157 95A06 879 919 843 750 571 A20 1101 873 597 501 391 A07 1129 965 750 585415 A08 1213 958 764 656 563 A09 1365 1170 896 742 600

An in vitro thrombin generation assay based on the cleavage of Z-GGR-AMCto release the fluorophore AMC was developed using normal human plasma,i.e. the Defined Dual-Pathway Thrombin Generation Assay. Tissue factordependency of peak thrombin generation and thrombin peak time wascharacterised in a composition containing a fixed concentration ofphospholipid (namely 3.2 μM). Phospholipid dependency was characterisedin a composition containing a fixed concentration of tissue factor(namely 7.2 pM). Peak time (time to peak thrombin generation) wasdependent on the concentration of phospholipid or tissue factor. Thefinal version of this assay is as described in the specific description,in which 10 μl reagent C high containing (32 μM phospholipid and 71.6 pMtissue factor) is used in a total volume of 100 μl.

Further studies were conducted using 3.2 μM phospholipid and 7.2 pMtissue factor in FVIII deficient or inhibited plasma, to characterisethe effect on peak thrombin generation and thrombin peak time of variouscoagulation factor preparations. These studies provided a basis fromwhich to compare the efficacy of the compounds in the assay. Briefly,rFVIII (Recombinate® FVIII from Baxter) was tested at 0, 5, 10, 20, 40and 80 mU/ml in FVIII deficient plasma. FEIBA was tested at 0, 8, 16,31, 63 and 125 mU/ml in FVIII inhibited plasma. FVIIa was tested at 0,0.1, 0.4, 1.6, 6.3 and 25 nM in FVIII inhibited plasma. Results areshown in FIG. 1. For recombinant FVIII (Recombinate®) in FVIII deficientplasma and for both FEIBA and FVIIa in FVIII-immuno inhibited plasma aconcentration dependent improvement of thrombin generation parameters isobserved. Peak thrombin increased and both the lag time and the thrombinpeak time decrease.

The compounds were tested in this Defined Dual-Pathway ThrombinGeneration Assay (DDPTGA) using reagent C high (Technoclone) fortriggering thrombin generation. Results are given in the table below.Even though this assay is less sensitive for FVIII-like activity thanthe Defined Intrinsic Thrombin Generation Assay (DITGA), severalcompounds possessed detectable activity.

TABLE 10 Summary results DDPTGA DDPTGA FVIII deficient FVIII inhibitedplasma plasma Peak Peak Peak Peak IIa Time Peak IIa Time IIa FVIII FEIBAFEIBA FVIIa FVIIa Peak Time EU Pep- Conc EU EU EU EU FVIII EU (mU/ tide[μM] (mU/ml) (mU/ml) (nM) (nM) (mU/ml) ml) A02 50 10.2 BLS 0.4 BLS BLSBLS A03 100 BLS BLS BLS BLS BLS BLS A03 50 BLS BLS BLS BLS BLS BLS A05100 BLS BLS BLS BLS BLS BLS A05 50 BLS BLS BLS BLS BLS BLS A08 50 9.1BLS 0.4 BLS BLS BLS A09 50 BLS 8.6 BLS BLS 27.2 BLS A09 25 7.9 BLS 0.2BLS 15.6 BLS A18 100 BLS BLS BLS BLS 17.1 BLS A18 50 BLS BLS BLS BLS BLSBLS A01 90 BLS BLS BLS BLS BLS BLS A01 50 BLS BLS BLS BLS BLS BLS A16100 BLS BLS BLS BLS BLS BLS A16 50 BLS BLS BLS BLS BLS BLS B03 100 BLS12.3 BLS 0.6 7.5 5.3 B03 40 BLS 7.2 BLS 0.2 8.7 4.8 B04 100 BLS 13.3 BLS0.7 1.6 5.0 B04 40 BLS 12.8 BLS 0.6 7.3 4.8 B04 16 BLS BLS BLS 0.1 4.14.4 A07 100 BLS BLS BLS 0.1 BLS BLS A07 50 BLS BLS BLS BLS BLS BLS A15100 BLS 13.6 BLS 0.7 12.7 5.0 A15 50 BLS BLS BLS BLS 14.4 4.3 A06 100BLS BLS 0.4 BLS BLS BLS A06 50 BLS BLS BLS BLS BLS BLS A14 100 BLS BLSBLS BLS BLS BLS A14 50 BLS BLS BLS BLS BLS BLS A17 100 BLS BLS 0.1 BLS5.9 BLS A17 50 BLS BLS BLS BLS 5.1 BLS

“Peak IIa” is the amount of thrombin generated at the peak of thethrombin generation curve. “Peak time” is the time from start of thethrombin generation reaction to when the maximum amount is generated.BLS=below lowest standard.

Thrombin is still generated in this assay even in the absence of addedpeptide. Thus, where Peak IIa is “BLS” at a particular peptideconcentration, there is still a thrombin peak, but it is lower than thatachieved by the lowest concentration of standard, which is 5 mU/mlFVIII, 8 mU/ml FEIBA or 0.1 nM FVII. Similarly, when peak time is “BLS”,the time to peak thrombin generation is greater than the peak timeachieved by the lowest concentration of standard. A peptide can have asignificant effect on peak time but not peak IIa, or vice versa.However, it is preferred that a peptide has an effect on both peak timeand peak IIa. B03, B04 and A15 positively affected both aspects ofthrombin generation. In the case of some peptides, concentrationdependency of an effect on thrombin generation was not seen at highpeptide concentration, which might be explained by non-specificinteractions.

Example 3: Testing of Compounds in Thrombin Generation Assays withSeveral Depleted Plasmas

The in vitro thrombin generation assay based on the cleavage ofZ-GGR-AMC to release the fluorophore AMC, described in the specificdescription, i.e. the Defined Dual-Pathway Thrombin Generation Assay wasused to characterise the effect of the compounds in several depletedhuman plasmas. In these experiments, each 100 μl reaction contained 10μl reagent B, which comprises phospholipid vesiclesPhosphatidylcholine/Phosphatidylserine 80%/20% (3.2 μM) and 17.9 pMrecombinant human tissue factor. 10 μl peptide dilution, 40 μl TGAsubstrate and 40 μl plasma were used as described in the specificdescription.

The plasmas used in the experiments were fresh frozen and were deficientin Factor V, Factor VII/VIIa, Factor VIII, Factor X or Factor XI (GeorgeKing Bio-Medical, Inc.). Residual coagulation factor levels of deficientplasmas were specified as less than 1%.

For each depleted plasma used in the experiments, compounds were testedat two concentrations, namely 50 μM and 80, 90 or 100 μM. A negativecontrol was used, in which no test compound was included. Results aresummarised in the table below.

TABLE 11 Summary results of effect of compounds on thrombin generationin various depleted plasmas De- Compounds pleted Con- plasma trol A01A02 A05 B03 A06 A07 A08 A09 FV − − − − − + − − FVII − + + + + + + +FVIII − + + + + + + + + FX − − + + + + + + FXI − + + + + + + +Stimulation of thrombin generation: “+” means stimulates; “−” means doesnot stimulate. In control experiments, no peptide was included.

All depleted plasmas tested showed no or very low thrombin generation inthe absence of peptides, indicating that at the tissue factorconcentration used the interplay of all coagulation factors is importantfor thrombin generation. Several peptides stimulated thrombin generationin all zymogen depleted plasmas (FVII, FX or FXI) whereas thrombingeneration in FV depleted plasmas is low, indicating that the commonpathway is important for peptide stimulated thrombin generation.

Example 4: Activity of Compounds in Defined Fibrin Deposition Assay

Various peptides were tested for the ability to stimulate fibrindeposition in the Defined Fibrin Deposition Assay as described in thespecific description. Results are shown in the table below.

TABLE 12 Compound characterization in the Defined Fibrin DepositionAssay Compound Concentration (μM) FEIBA EU (mU/ml) A01 100 145 A01 50127 A01 25 80 B01 100 94 B01 50 47 B01 25 31 A05 50 219 A05 25 193 A0510 119 B03 25 325 B03 12.5 291 B03 6.3 264 A06 25 168 A06 12.5 232 A066.3 251 A07 50 199 A07 25 246 A07 12.5 268

All test compounds shortened the clotting time and fibrin formation ofFVIII inhibited plasma. In combination with the thrombin generationexperiments this confirms the procoagulant activities of the testcompounds. Most compounds acted in a concentration dependent manner,although a small number had reduced activity at higher concentrations,which may be due to non-specific interactions.

Example 5: In Vitro Assays for the Characterisation of Compounds

Compounds are characterised not only for activity in the thrombingeneration assays but also for pharmacokinetics, solubility, HERGinhibition and molecular weight.

Pharmacokinetic (PK) Studies

PK studies are required for the design and interpretation of in vivoefficacy studies. Plasma protein binding, plasma stability andmicrosomal stability are all included in this category.

1. Plasma Protein Binding

The extent of compound binding to human plasma (Bioreclamation,Hicksville, N.Y.), mouse plasma (Lampire Laboratory, Pipersville, Pa.)or mouse serum albumin (Sigma, St. Louis, Mo.), referred to as matrices,was determined in a 96-well micro-equilibrium dialysis block system(HDT-96; HTDialysis, LLC, Gales Ferry, Conn.). Briefly, each unit of thesystem comprises a donor chamber and a receiver chamber separated by asemi-permeable membrane. The principle of the experiment is thatproteins (and compound bound to the proteins) are retained in the donorchamber and cannot cross the membrane. Free compound can diffuse betweenboth chambers via the membrane and reaches equilibrium during theexperiment. In these experiments, the semi-permeable membrane was madeof regenerated cellulose and had a molecular weight cut-off of 12-14 kD(cat. no 1101, HTDialysis, LLC).

A protease inhibitor cocktail (P2714-1BTL), purchased from Sigma, wasincluded in the assay to inhibit proteolysis of test compounds. It wasfreshly prepared at 50× stock solution in distilled water. Mouse serumalbumin was freshly prepared in phosphate buffered saline (PBS) at 40g/L. The PBS was purchased from Invitrogen (Carlsbad, Calif.), and itwas adjusted to a pH of 7.4 prior to use. Plasmas were used withoutdilution. The protease inhibitor stock solution was added to each matrix(i.e. mouse serum albumin in PBS) at a final 1× concentration. Stocksolutions of each test compound were prepared in DMSO with the controlcompound, warfarin. Warfarin, which is a high protein-binding compound,was included in each stock solution to ensure the integrity of themembrane during the experiment. An aliquot of the stock solution wasadded to each matrix to yield a final concentration of 5 μM of the testcompound and 10 μM of warfarin. The final concentration of DMSO was0.72% (v/v). The dilution of the matrices by the addition of the othercomponents was negligible (less than 4%). The membrane strips werehydrated in distilled water for 1 hour; the membrane was soaked in 30%ethanol aqueous solution for 20 minutes, and then the membrane wasrinsed twice with distilled water. After the rinse, the membrane wasplaced in PBS and was ready for use. The assembly of the dialysis blockfollowed the manufacturer's protocol. After the assembly, an aliquot of150 μl of each matrix/test compound was added to a separate donorchamber and 150 μl of PBS was added to the corresponding receiverchamber on the other side of the membrane. The remainder of eachmatrix/test compound was stored at −80° C. for further analysis. Theconcentrations of the test compounds and warfarin in these matrices weremeasured and the values were used in the recovery calculations. The96-well dialysis block was then placed in an enclosed, heated rocker,which was pre-warmed to 37° C., and allowed to incubate for 6 hours.After the incubation, both sides were sampled. The concentrations of thetest compounds, as well as warfarin, were measured by LC/MS/MS analysis.

The recovery and protein binding values were calculated as follows:% Recovery=[(Conc. in Donor+Conc. in Receiver)/(Measured Conc. inMatrix)]×100%  (1)% Bound=[(Conc. in Donor−Conc. in Receiver)/(Conc. in Donor)]×100%  (2)

“% Recovery” is a measure of how much of the compound added to thematrix is recoverable from the donor and receiver chambers. Whererecovery is less than 100%, a proportion of the compound may have boundto the membrane or the plastic surfaces of the chambers or it may havedegraded. “% Bound” is a measure of how much of the compound has boundto the matrix and is therefore unable to equilibrate between donor andreceiver chambers.

Results are shown for A01 and warfarin (control) in the tables below.

TABLE 13 Protein Binding of A01 in Tested Matrices A01 Donor Conc. inReceiver Concentrations Matrix Concentrations (μM) (μM) Average Average% Matrix (μM) Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 % Bound Recovery Human1.69 <0.025 <0.025 <0.025 1.47 1.57 1.45 >98.3 >88.6 plasma Mouse 4.48<0.025 <0.025 <0.025 3.18 3.43 3.24 >99.2 >73.3 plasma Mouse 1.42 <0.005<0.005 <0.005 1.50 1.43 1.47 >99.7 >103 serum albumin

TABLE 14 Protein Binding of warfarin in Tested Matrices Warfarin DonorConc. in Receiver Concentrations Average Average % Matrix Concentrations(μM) (μM) % Bound Recovery Matrix (μM) Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep3 (StDev) (StDev) Human 9.88 0.0749 0.0765 0.0791 9.86 9.43 9.91 99.299.3 (2.68) plasma (0.03) Mouse 10.1 0.457 0.403 0.400 8.37 8.72 8.5095.1 88.6 (1.53) plasma (0.462) Mouse 9.61 0.365 0.354 0.347 9.78 9.148.34 96.1 98.3 (7.60) serum (0.218) albumin

2. Plasma Stability

Half-life of compounds in human or mouse plasma, or percentage ofcompound remaining after an incubation in human or mouse plasma, isdetermined as follows. In the experimental procedure, test compoundconcentrations are 5 μM, prepared from test compound stock solutions of10 mM in DMSO. Propantheline is used as a standard. To prepare testssamples, a 1/20 dilution of the test compound stock solution in DMSO isprepared in 50% acetonitrile/50% H₂O, and this is then diluted 1/100 inpre-warmed (37° C.) plasma (5 μl compound [ 1/20 dilution]+495 μlplasma) in a 1.5 ml Eppendorf-tube. The standard compound 2 mMpropantheline is diluted ¼ in DMSO and subsequently 1/100 in pre-warmedplasma (5 μl compound [¼ dilution]+495 μl plasma) in a 1.5 mlEppendorf-tube. All of the samples are incubated in a water bath at 37°C. 500 μl acetonitrile is added immediately after compounds, orpropantheline standard, have been mixed with plasma (designated as t=0min). After a chosen duration of incubation (generally at t=60 min) eachsample is mixed with a further 500 μl acetonitrile. The samples aremixed on a vortex mixer for 30 s and placed on ice for 10 min andcollected for centrifugation. Samples are centrifuged at 20 000 g for 10min at 4° C. 500 μl supernatant is transferred into a new 1.5 mlEppendorf tube and an equal volume of acetonitrile is added. The sampleis mixed again for 30 s using a vortex mixer. After a secondcentrifugation step (20 000 g, 10 min, 4° C.) 250 μl of the supernatantis transferred into HPLC glass vials for HPLC-MS analysis. Conditionsfor performing HPLC are as follows: Injection-volume is set to 20 μl.Temperature is set to 25° C. A linear gradient from 95:5 to 5:95water:acetonitrile both containing 0.05% trifluoroacetic acid (TFA)(v/v) is applied at a flow rate of 0.3 ml/min for 10 min. ThePDA-detector is scanning from 210-400 nm. The ion-trap is equipped withan ESI-source with temperature at 280° C., mass-scanning is done in fullscan-mode from 50-2000 amu followed by dynamic exclusion MS²-experimentwith 1.5 V collision energy (105 as min. count of parent ion).Percent-stability is calculated from area-under-curve (AUC) ratiomonitoring the protonated molecular-mass ion of the target compound inthe total-ion-current (tic) in full-scan mode at 60 min incubation time(or time of choice) vs. 0 min incubation time.

Results are shown in the table below. Decreases in compoundconcentration over time might be due to proteolytic degradation and/orchemical modification.

TABLE 15 Plasma stability of compounds Peptide % remaining in humanplasma (30 min) A01 58 A19 99 A07 117 A20 95 A06 98 A02 84 A03 92 A08 70A09 67 B07 89 B06 93 B05 112 A05 93

3. Microsomal Stability

Tests were used to determine the stability of compounds in microsomalpreparations of human or animal origin. The microsomal stability ismeasured either in assays provided by Cerep (France, Catalog ref.900-8h) or by the protocol described below. Compound solutions of 10mM/5 mM (test compound, standards verapamil, imipramine, andterfenadine) are prepared in 100% DMSO. They are diluted by distilledH₂O/MeOH resulting in a final concentration of 1 μM in the assay, withless then 0.4% DMSO (v/v) in the final mixture. The mastermix for thestability assay is prepared in a 10 ml Falcontube (total volume 4.4 ml):3414 μl distilled water, 440 μl 500 mM NaPO₄-buffer pH 7.4, 440 μl NADP(10 mM), 22 μl Glc-6-P (1 M), 17.6 μl Glc-6-P-DH of a 1 U/ml solution,66 μl liver microsomes (rat or mouse, final concentration in the assay300 μg/ml). The mastermix is preincubated at 37° C. for 10 minutes inthe water bath. 5 μl of 60 μM compound solution is added per well in a96-well-U-Plate (PP-Nunc) together with 300 μl of reaction mixture(pre-incubated mastermix). All wells must be carefully mixed to ensure ahomogenous suspension before the next steps. 75 μl samples (duplicates)at t=0 minutes are taken for each compound. The plate is sealed andreturned to the water bath/thermomixer for 30 minutes. The testcompounds/standards are extracted by addition of 200 μl methanol, alsoincluding an internal standard. The internal standard is “Pep770”(Jerini A G, Berlin, Germany) and is used at a final concentration of6.25 ng/ml. The samples are centrifuged at 1300 g for 10 min at 4° C.200 μl of the supernatant is transferred into a 96-well plate with 10 μlDMSO per well. Compound stability is measured by HPLC-MS analysis(triplicates). The same procedure is repeated after 30 min. The ratio ofthe mean “AUC t=0 min” and “AUC t=30 min” is calculated and thepercentage of the amount of remaining compound after 30 min isdetermined. The signal to noise ratio for all peaks must be 5:1 orbetter. The ratio AUC_(analyte):AUC_(standard) at the differenttimepoints must be used. The calculated stability for the controlcompound must fall in a certain range to validate the assay.

Results are shown in the table below.

TABLE 16 Stability of compounds in mouse microsomes Peptide Stabilityafter 30 min [%], duplicates A01 27/32 A02 44/45 A06 51/47 A07 39/28 A0922/23 Terfenadine 63/68

Solubility

Aqueous solubility is measured in PBS at pH 7.4 either in an assayprovided by Cerep (France, Catalog ref. 900-11a) or according to thefollowing protocol. The aim of this procedure is to determine thesolubility of a drug candidate (analyte) in a buffer, by estimating thesaturation concentration of the candidate in the buffer using HPLC. Aknown concentration of the candidate in an organic solvent is used as astandard. A stock solution of the test compound in DMSO must be preparedas the initial step. Depending on the maximum solubility of thecompound, a concentration of 50 mM in DMSO should be reached. DMSOstock-solutions are diluted to a final concentration of 50 μM with DMSO(100% reference-solution) and buffer (test-solution) to provide aminimum volume of 500 μL of each. Both solutions are shaken at 25° C. at950 rpm in an Eppendorf “thermomixer comfort” for at least 60 min. Thesuspension is centrifuged at 22 330 g for at least two min and 100 μL ofthe supernatant is transferred into polypropylene-inserts placed inglass vials and closed by snap-ring caps. Alternatively the solutionscan be prepared in microtiter plates with half of the previouslydescribed starting solvent volume. For the determination of thesolubility all samples are analyzed by HPLC in triplicate. Injectionvolume is at least 10 μL. The obtained data are analyzed by“Chemstation-software” (Agilent, Waldbronn, Germany). The peaks from theanalyses of the organic solutions are integrated and the arithmetic meanis reported as “AUC 1” (reference area of known amount injected at theHPLC). The same procedure is applied to the spectra obtained from theanalyses of the buffer solution to give “AUC 2” (area of the unknownamount of compound dissolved in buffer). In general the AUC must begreater then 20 area units and signal to noise (height of the peak) mustbe better than 3. The ratio of the mean “AUC 2” and “AUC 1” iscalculated and thus the percentage of the dissolved amount of compoundin buffer is obtained and solubility can be reported in μM.

Results are shown in the table below.

TABLE 17 Solubility of compounds in PBS Compound Solubility in PBS [μM]A09 63 B03 114 B07 195 B06 48 B05 174 A01 9 A08 11 A05 6 A19 14 A07 165A20 166 A06 164 A02 35 A03 110 A16 >200

HERG Inhibition

QT prolongation is assessed by HERG inhibition measured by patch-clamptechniques or Rb⁺ efflux.

The Rb+ efflux method (Cerep, France, Catalog. Ref. 900-36rb) is usedfor initial screening. For the Rb+ efflux assay, the reference compound(Astemizole) was tested concurrently with the test compounds in order toassess the assay suitability. It was tested at 10 μM and the data werecompared with historical values determined at Cerep.

For rigorous characterization of HERG inhibition the patch clamp assayis applied (Cerep, France, Catalog ref. 900-36). The general potencyranking system is adopted from Roche et al., 2002, Chem Bio Chem3:455-459. To ensure that no change in the sensitivity of the assay hasoccurred, separate experiments conducted on the same (clone) batch ofcells using 10 nM E-4031 (Wako, cat. no. 052-06523) yielded results(56.7±1.8% inhibition, Mean±SEM, n=3) comparable to historicallyobtained data (58.4±2.0% inhibition, Mean±SEM, n=3) at Cerep. The testcompounds (10 mM stock solutions) were dissolved in dimethylsulfoxide(DMSO). The solutions at 1 μM contained 0.01% DMSO. Bath solutionscontaining up to 1% DMSO have no significant effect on the HERG-encodedtail currents.

Screening of several compounds by the Rb+ efflux method at 10 μMindicated no inhibition of HERG channel activity. In the more sensitivepatch clamp assay compounds A01, A05 and A16 can be classified aslow-potency HERG-channel blockers whereas B03 was identified as a highpotency HERG-channel blocker. Results are provided in the table below.

TABLE 18 HERG inhibition results HERG inhibition patch-clamp HERGinhibition (% Inhibition of Tail Rb+ efflux Compound Current at 1 μM) (%Inhibition at 10 μM) A01 16.5 A02 −3.3 A05 14.5 B03 84.5 −2.1 A06 −6.4A07 −3.4 A09 −1.6 A16 22.2 Astemizole 73.5

Molecular Weight

The molecular weight is defined as the theoretical mass of the monomericmolecule exclusive of any counter ions or adducts. The molecular weightsof the compounds are indicated in the table below.

TABLE 19 Molecular weights of compounds Compound Molecular weight(g/mol) A09 2175 B03 1439 B07 1423 B06 1849 B05 1763 A01 2087 A08 2175A05 1311 A19 1410 A07 1743 A20 1742 A06 1741 A02 2028 A03 2074 A16 2087

Example 6: ADME-Tox

ADME-Tox analyses of various compounds were performed as described inExample 5. Summary results are shown in the table below.

TABLE 20 Summary ADME-Tox data A01 A02 A05 B03 A06 A07 A09 A16 A17 Aque-9 35 6 114 164 165 63 220 2000 ous solu- bility Prote- 58 84 94 98 11767 olytic stability (human) Micro- 30 45 49 34 23 somal stability HERG17 15 85 22 channel

Briefly, aqueous solubility was tested in PBS pH 7.4. Results are givenin μM. Proteolytic stability was tested in human plasma for 30 minutes.Results for each are given as % stability. Microsomal stability wastested in a mouse microsomal preparation for 30 minutes. Results aregiven as % stability. HERG channel inhibition was tested using the patchclamp method in which the peptide or peptide derivative at 1 μM and aregiven as % inhibition.

Example 7: Animal Models

The following assays are performed in animals.

1. Acute Toxicology

Toxicology studies involved monitoring of attitude changes due to toxiceffects immediately after application and twice daily; monitoring ofbody weight; histopathology of brain, heart, kidney, liver, lung.Experiments were performed in C57Bl/6 mice.

2. Pharmacokinetics

Pharmacokinetics of compounds were tested in C57Bl/6 mice or Wistar ratsat 1-30 mg/kg. Compound concentrations in the blood stream weremonitored at appropriate intervals using LC-MS.

3. Circulation Analysis

Blood pressure and heart rate were monitored and electrocardiogram takenin C57Bl/6 mice.

4. Animal Disease Model

A tail clip model was used in FVIII −/− (E17) mice, FIX −/− mice andC57Bl/6 control mice. Parameters quantified were total blood loss,bleeding time, bleeding rate and survival.

Example 8: Acute Toxicology

C57Bl/6 mice weighing 18-20 g were administered with compounds in asuitable vehicle i.v. in the tail vein or i.p. or s.c at 10 ml/kg.Quantities of the compounds administered were in the range of 0.075 to125 mg/kg (i.v.), 15-125 mg/kg (i.p.) and 125 mg/kg (s.c.). There werefour mice per group. Attitude changes due to toxic effects weremonitored immediately after administration of the compound and for 60minutes thereafter. Body weight was monitored for five days afteradministration. At day 5 after administration, mice were culled andautopsy performed. Brain, heart, kidney, liver, lung and spleen werebiopsied. Results are described below.

TABLE 21 Attitude changes due to toxic effects at different compounddoses Max dose (mg/kg) within toxicity category No detected Compoundroute toxicity Some toxicity Severe toxicity A01 i.v. 20 i.p. 20 A02i.v. 25 i.p. 25 A05 i.v. 1.5 15 i.p. 15 B03 i.v. 1.5 15 i.p. 15 A06 i.v.1 5 25 i.p. 25 A07 i.v. 1 5 25 i.p. 25 A09 i.v. 15 i.p. 15 A16 i.v. 82i.p. 82 A17 i.v. 125 i.p. 125 s.c. 125

In the above table, the maximum dose tested giving rise to no detectedtoxicity, some toxicity or severe toxicity over the period of 60 minutesfollowing administration is reported. “No detected toxicity” indicatesno acute toxic observations. “Some toxicity” indicates that ataxy orcatalepsy were recorded, but no animals died. “Severe toxicity”indicates that one of the animals died within one hour of compoundapplication.

In summary, most of the compounds were tolerated well. Doses ofcompounds which resulted in severe toxicity when administered by aparticular route were not tested in pharmacokinetics, circulationanalysis or animal disease models by that route.

For most of the compounds, even at the highest dose, no macroscopicpathological findings were observed in biopsy samples collected at fivedays from surviving mice, indicating that the compounds were toleratedwell. The only pathological changes identified in any animal were minorabnormalities in the liver, kidney, lung or heart. These werespontaneous observations in single animals probably due to noncompound-related minor infections or due to culling.

For each compound tested, no effect on average body weight of survivingmice, indicative of a negative response, was noted.

Example 9: Pharmacokinetics of Compounds

Pharmacokinetic studies were performed to monitor compoundconcentrations in plasma following i.v., i.p. or s.c. administration.Studies were conducted in C57Bl/6 mice weighing approximately 20 g.

For each peptide, the same formulations were used for all administrationroutes and were as follows: A01 was formulated in 5% DMSO, 5% CremophorEL (Sigma-Aldrich), 0.5% Tween 80; A02 and A09 were each formulated in5% DMSO, 30% PEG 400 (polyethylene glycol) 50 mM sodium phosphate pH7.4; A05 was formulated in 5% DMSO, 20 mM glycine pH 9.0; A06 and A07were each formulated in 5% DMSO, 0.9% NaCl, 50 mM sodium phosphate pH7.4.

Peptide concentrations in the plasma were analysed by HPLC-MS on aSurveyor HPLC combined with mass spectrometer LCQ classic or Advantage(all Thermo Electron, US) equipped with an ESI-source. All HPLCexperiments were carried out on a Phenomenex C-18 Luna column (50 mm×2.0mm, 5 μl injection volume) using a linear gradient: eluent A 0.05%trifluoracetic acid (TFA) in water; eluent B 0.05% TFA in acetonitrile;flow rate 0.3 mL/min in 10 min. UV spectra were recorded by the PDA from220 to 400 nm. The internal standard is prepared as a 0.1 μg/ml solutionin 100% methanol. 50 μl plasma and 50 μl internal standard are mixed.100 μl methanol is added and mixed thoroughly. After 30 min incubationon ice the vial is centrifuged for 15 min at 4° C. (20820 g). 150 μl ofthe supernatant are transferred into the HPLC vial

Results following i.v. or i.p. administration are shown in the tablebelow. In brief, peptide clearance from plasma following i.v.administration followed a roughly logarithmic course. Following i.p.administration, Cmax was reached at between 40 and 60 minutes. Therethen followed a decrease in compound concentration. This profile istypical for i.p. or s.c. administration.

TABLE 22 Data obtained in pharmacokinetics analyses Peptideconcentration in plasma (μg/ml) at time intervals after admin. [min]i.v. 5 10 15 20 25 30 A01 0.673 0.352 0.454 0.145 0.282 0.115 A02 11.6257.260 3.182 2.738 1.436 1.308 A05 2.605 2.039 0.173 0.101 0.023 — A0649.963 32.610 20.981 15.593 11.267 7.787 A07 12.512 7.796 5.482 2.1323.309 2.218 A09 0.350 0.184 0.109 0.119 0.064 0.080 [min] i.p. 5 20 4060 80 100 A02 0.273 0.315 0.289 0.612 0.099 0.176 A05 0.119 0.165 0.1850.130 0.049 0.050 A06 6.442 7.773 0.547 5.800 4.057 3.024 A07 2.4442.627 3.180 3.869 2.202 1.678 A09 0.034 0.067 0.060 0.129 0.074 0.068

Example 10: Circulation Analysis Following Administration of A01

Mean arterial blood pressure and heart rate were monitored andelectrocardiogram taken in three groups of three male and three femaleC57Bl/6 mice, each weighing about 20 g. The groups were assigned to“control” receiving 10 ml/kg NaCl i.v.; “vehicle” receiving 10 ml/kgi.v.; or “compound” receiving A01 in vehicle at 20 mg/kg i.v. “Vehicle”was DMSO 5%, Cremophor EL (Sigma-Aldrich) 5%, Tween 80 0.05% in waterfor injection.

For each mouse, a catheter filled with saline/heparin was fixed to theaorta carotis. The catheter was linked via a transducer to a bloodpressure Plugsys-module (Hugo Sachs Electronik-Harvard Apparatus GmbH,Germany (HSE)). ECG electrodes were implanted s.c. and were linked via aECG Plugsys-module (HSE) to a PC. Heart rate was calculated from theECG. After a period of at least ten minutes to stabilize circulationparameters, saline, vehicle or compound was administered as appropriatevia a catheter connected to the vena jugularis. Circulation parameterswere monitored and recorded for 60 minutes after administration. Foreach animal, the time course in mean arterial blood pressure and heartrate within the observation period after study drug administration wasestimated using the area under the curve (AUC) using the lineartrapezoidal rule. The individual AUCs (A01 20 mg/kg i.v.) were comparedwith those of vehicle (10 mL/kg i.v.) and saline (10 mL/kg i.v.). Thenull hypothesis (no differences between compound and vehicle or saline)were assessed using the exact Wilcoxon rank sum test. Unadjusted andadjusted two-sided p-values for multiple comparisons were calculated.Adjustment for multiplicity was performed by using the Bonferroni-Holmmethod. The level of significance was set to 5%. All statisticalanalyses were performed with R Version 2.4.0. The null hypothesis of nodifference was tested against the two-sided alternative. Results areshown in the table below.

TABLE 23 Statistical results of circulation analysis Unadjusted two-Adjusted two- Parameter Comparison sided p-value sided p-value Meanarterial BP A01 vs. saline 0.5887 1.0000 A01 vs. vehicle 1.0000 1.0000Heart rate A01 vs. saline 0.6991 0.6991 A01 vs. vehicle 0.2403 0.4805

There were no statistically significant (at the 5% level) differences inAUC of mean arterial blood pressure within 60 min after study drugadministration between A01 20 mg/kg i.v. and saline 10 ml/kg i.v. aswell as between A01 20 mg/kg i.v. and vehicle 10 ml/kg i.v. There wereno statistically significant (at the 5% level) differences in AUC inheart rate within 60 min after study drug administration between A01 20mg/kg i.v. and saline 10 ml/kg i.v. as well as between A01 20 mg/kg i.v.and vehicle 10 ml/kg i.v.

Example 11: Animal Disease Model—Control Experiments

Experiments were performed to develop a mouse tail clip assay tocharacterise bleeding parameters in FVIII (E17)−/−, FIX−/− (Lin H FBlood 1997; 90: 3962-6) and wild-type C57Bl/6 mice and their response tocoagulation factor preparations.

Coagulation factor preparations tested were Advate® and Immunine®.Advate® is a rFVIII preparation (Baxter AG, Austria). Immunine® is apurified plasma FIX preparation (Baxter AG, Austria).

Blood loss was monitored in the tail clip assay as described in thespecific description for 62 minutes after tail clip. FVIII −/− mice wereadministered with rFVIII (Advate®) at 25, 50 or 100 U/kg i.v. or withvehicle alone. The vehicle was Advate formulation buffer which is 38mg/ml mannitol, 10 mg/ml trehalose, 108 mEq/l sodium, 12 mM histidine,12 mM Tris, 1.9 mM calcium, 0.17 mg/ml Polysorbate-80, 0.1 mg/mlglutathione. As a control, C57Bl/6 mice were administered vehicle alone.Administration of rFVIII resulted in a dose-dependent reduction of bloodloss over the 62 minutes. Survival data for the experiment are shown inthe table below.

TABLE 24 Survival of FVIII −/− mice treated with Advate ® FVIII in tailclip experiment Survival Survival Survival Survival Dose Animals (%) (%)(%) (%) (U/kg) (n) 2 hours 4 hours 24 hours 48 hours Advate 100 10 10090 90 90 Advate 50 10 100 100 90 90 Advate 25 10 100 100 90 90 Vehicle —10 70 50 50 50 C57BI/6 — 16 100 100 100 100

Blood loss was monitored for 62 minutes after tail clip of FIX −/− miceadministered with Immunine® FIX at 50, 100 or 200 U/kg i.v. or withvehicle alone. As a control, C57Bl/6 mice were administered vehiclealone. Administration of FIX resulted in a dose-dependent reduction ofblood loss over the 62 minutes. Survival data for the experiment areshown in the table below.

TABLE 25 Survival of FIX −/− mice treated with Immunine ® FIX in tailclip experiment Survival Survival Survival Survival Dose Animals (%) (%)(%) (%) (U/kg) (n) 2 hours 4 hours 24 hours 48 hours Immunine 200 16 100100 100 100 Immunine 100 16 100 100 94 94 Immunine 50 16 94 69 63 63Vehicle — 16 44 19 6 0 C57BI/6 — 16 100 100 100 100

The data show that in the FVIII −/− model, Advate FVIII at 25-100 U/kgdose dependently improves bleeding parameters and survival. In the FIX−/− model, Immunine® FIX at 50-200 U/kg dose dependently improvesbleeding parameters and survival. Thus, the FVIII −/− model is anappropriate model to test coagulation FVIII activity of the leadcompounds. The FIX −/− model is an appropriate model to test coagulationFIX activity of the compounds.

Example 12: Animal Disease Models—Efficacy of A01

The effect of administered A01 on bleeding parameters and survival ofFVIII −/− mice was tested in the tail clip model described in thespecific description. Similar experiments were performed in FIX −/−mice.

Mean volume of blood loss following tail clip in a group of 8 male and 8female FVIII −/− mice administered 20 mg/kg of A01 i.v. five minutesbefore tail clip was significantly different (p<0.05) at most timepoints compared to mean volume of blood loss by a control group of miceadministered vehicle alone. Vehicle was 5% DMSO, 5% Cremophor EL, 0.05%Tween 80 in water for injection. At 52 and 62 minutes after tail clip,the difference was significant at p<0.01. Data are shown in FIG. 2 andthe table below. A log-rank test used to compare the survival curves ofmice in this experiment shows a statistically significant longersurvival with A01 20 mg/kg i.v. than with vehicle control(p-value=0.0028).

TABLE 26 Survival of FVIII −/− mice treated with A01 in tail clipexperiment Survival Survival Survival Survival Animals (%) (%) (%) (%)Treatment (n) 2 hours 4 hours 24 hours 48 hours A01 16 56 25 6 0 Vehicle16 6 6 0 0

The above experiment was repeated to provide an indication of itsreproducibility. Results are shown in the table below. Althoughvariability is observed within these two independently performedexperiments animals treated with A01 bleed less and survive longer.

TABLE 27 Survival of FVIII −/− mice treated with A01 in tail clipexperiments Survival Survival Animals (%) (%) Survival (%) Survival (%)Exp (n) 2 hours 3 hours 4 hours 24 hours A01 1 16 56 44 25 6 Vehicle 116 6 6 6 0 A01 2 16 63 38 31 13 Vehicle 2 16 44 25 25 0

Further data were obtained using the same model, although the tail clipwas 1 cm rather than 0.5 cm, and mice were grouped according to gender.Agents were administered i.v. In this experiment, it appeared that A01was more effective in female than male mice. Results are shown in thetable below.

TABLE 28 Gender effects of A01 in FVIII −/− mouse tail snip experimentNo. of Survival 2 Survival 24 Compound animals (n) hours (%) hours (%)A01 formulation buffer 20 30 0 A01 formulation buffer 10 female 50 0 A01formulation buffer 10 male 10 0 A01 20 mg/kg 10 female 80 10 A01 20mg/kg 10 male 20 0 Advate formulation buffer 10 10 0 Advate 200 IU/kg 1090 90 Advate 100 IU/kg 10 70 50

Groups contained equal numbers of male and female mice, unless otherwisestated.

Mean volume of blood loss following tail clip in a group of 16 FIX −/−mice administered 20 mg/kg of A01 i.v. five minutes before tail clip wasnot significantly different (p<0.05) at any time points compared to meanvolume of blood loss by a control group of mice administered vehiclealone. There was no significant difference in survival of miceadministered A01 and mice administered vehicle alone.

These results demonstrate that A01 can at least partially compensate forthe lack of FVIII in FVIII−/− mice by reducing blood loss and increasingsurvival following tail clip, but has no effect in FIX−/− mice. A01 isregarded as the most preferred peptide because it has demonstratedefficacy in a hemophilia model.

Example 13: Results of Compound Testing in FVIII−/− Mouse Tail ClipModel

A01 was further tested by i.p. administration in the FVIII −/− tail clipmodel using a 0.5 cm tail clip. Data are summarised in the table below.

TABLE 29 Survival of FVIII −/− mice treated with A01 in tail clipexperiment Total blood Survival loss Survival Survival 24 Com- TreatmentAnimals (% of 2 hours 4 hours hours pound dose (n) Vehicle) (%) (%) (%)A01 20 mg/kg 8 72 75 25 25 i.p. female A01 20 mg/kg 8 83 38 25 25 i.p.male Vehicle 10 ml/kg 8 13 0 0 control i.p. female Vehicle 10 ml/kg 8 1313 13 control i.p. male

Female mice administered with A01 20 mg/kg had a statisticallysignificant (at the 5% level) longer survival than female miceadministered with vehicle 10 ml/kg i.p. (two-sided p-value p=0.0073;log-rank test). There was no statistically significant difference (atthe 5% level) in survival curves between male mice administered with A0120 mg/kg i.p. and male mice administered with vehicle 10 ml/kg i.p. Inthis experiment, the males in the control group seemed to survive betterthan the females in the control group.

Example 14: Summary of Experiments for Characterising Lead Compounds

The following compounds possess activity in the Defined IntrinsicThrombin Generation Assay: A01, A03, A05, A19, B01, A02, B03, B05, B06,A06, A20, A07, A08, A09 and B07. Of these, A03, A02, B03, A08 and A09have a thrombin generation activity at 100 μM of at least 1200 mU/mLFEIBA.

The following compounds possess activity in the Defined Dual-PathwayThrombin Generation Assay: A02, A03, A08, A09, A18, B03, B04, A07, A15,A06 and A17. Of these, A09 had a peak IIa activity at 50 μM of at least10 mU/mL FEIBA. B03, B04 and A15 had a peak IIa activity at 50 or 100 μMof at least 10 mU/mL FEIBA.

The following compounds possess activity in the Defined FibrinDeposition Assay: A01, 601, A05, B03, A06 and A07.

The following compounds have a stability of at least 50% followingincubation for 30 minutes in human plasma: A01, A19, A07, A20, A06, A02,A03, A08, A09, B07, B06, B05 and A05.

The following compounds have solubility in PBS pH 7.4 of at least 25 μM:A09, B03, B07, B06, B05, A07, A20, A06, A02, A03 and A16. Of these, B03,B07, B05, A07, A20, A06, A03 and A16 have a solubility in PBS pH 7.4 ofat least 100 μM.

A01, A05 and A16 were identified as low-potency HERG-channel blockers.

A01 was identified as possessing activity in the tail clip assay inFVIII−/− mice.

Example 15: Treatment of Hemophilia A in an Adult Human Subject

It is typical for hemophilia A patients to develop alloantibodyinhibitors to FVIII following high dose FVIII therapy. In a typicalscenario, the presence of such antibodies in serum prepared from thepatient's blood plasma is monitored by a clinician. When the titre ofthe antibody response becomes unacceptably high, such as about 5 BU, theclinician may decide to stop infusing the patient with FVIII, and startadministering a peptide of the invention, such as peptide A01.

The peptide may be formulated as a microparticle of about 10 μm diameterin an albumin shell, suspended in an aqueous medium, as described inU.S. Pat. No. 5,439,686. The patient may self administer the formulationby inhalation using a nebulizer. A daily or twice daily dose of 5 or 10mg may be inhaled. The clinician may test the partial thromboplastintime shortly after commencement of the peptide therapy, to confirmefficacy. Depending on the result, the dose could be varied accordingly.If it is necessary to substantially increase the dose, smallermicroparticles could be used, typically of about 5 μm diameter, and theycould be administered intravenously.

The invention claimed is:
 1. A method of treating a patient having adeficiency in FV, FVII, FVIII, FX and/or FXI, said method comprisingadministering to the patient a therapeutically effective amount of apeptide or peptide derivative comprising: (i) WDLYFEIVW (SEQ ID NO:322);(ii) a variant amino acid sequence comprising one, two, three or fouramino acid substitutions in WDLYFEIVW (SEQ ID NO:322), said variantamino acid sequence comprising X₁X₂X₃YX₄EX₅X₆X₇, wherein: X₁ is W, L orP; X₂ is D or S; X₃ is L or F; X₄ is F, Phg, L, Ebw, Pff, Thi, 1Ni, Hfe,Ece or Cha; X₅ is I or F; X₆ is S, V or G; and X₇ is W or L (SEQ IDNO:322); or (iii) a retro-inverso variant of the peptide or peptidederivative of either (i) or (ii)′ wherein said peptide or peptidederivative has procoagulant activity; and wherein said patient hasinhibitor antibodies against FV, FVII, FVIII, FX and/or FXI.
 2. A methodof treating a patient having a deficiency in FV, FVII, FVIII, FX and/orFXI, said method comprising administering to the patient atherapeutically effective amount of a peptide or peptide derivativecomprising: (i) WDLYFEIVW (SEQ ID NO:322); (ii) a variant amino acidsequence comprising one, two, three or four amino acid substitutions inWDLYFEIVW (SEQ ID NO:322), said variant amino acid sequence comprisingX₁X₂X₃YX₄EX₅X₆X₇, wherein: X₁ is W, L or P; X₂ is D or S; X₃ is L or F;X₄ is F, Phg, L, Ebw, Pff, Thi, 1Ni, Hfe, Ece or Cha; X₅ is I or F; X₆is S, V or G; and X₇ is W or L (SEQ ID NO:322); or (iii) a retro-inversovariant of the peptide or peptide derivative of either (i) or (ii)wherein said peptide or peptide derivative has procoagulant activity;and wherein said administering is selected from the group consisting ofintravenous, intraperitoneal, subcutaneous, nasal, buccal, oral, andpulmonary delivery.